Nucleic acids encoding chromophores/fluorophores and methods for using the same

ABSTRACT

Nucleic acid compositions encoding novel chromo/fluoroproteins and mutants thereof, as well as the encoded proteins, are provided. The subject proteins of interest are proteins that are colored and/or fluorescent, where this feature arises from the interaction of two or more residues of the protein. The subject proteins are further characterized in that they are either obtained from non-bioluminescent  Cnidarian , e.g., Anthozoan, species or are obtained from non-Pennatulacean (sea pen) species. Specific proteins of interest include proteins obtained from the following specific Anthozoan species:  Anemonia majano  (NFP-1),  Clavularia  sp. (NFP-2),  Zoanthus  sp. (NFP-3 &amp; NFP-4),  Discosoma striata  (NFP-5),  Discosoma  sp. “red”(NFP-6),  Anemonia sulcata  (NFP-7),  Discosoma  sp “green” (NFP-8), and  Discosoma  sp. “magenta” (NFP-9). Also interest are proteins that are substantially similar to, or mutants of, the above specific proteins. Also provided are fragments of the nucleic acids and the peptides encoded thereby, as well as antibodies to the subject proteins and transgenic cells and organisms. The subject protein and nucleic acid compositions find use in a variety of different applications. Finally, kits for use in such applications, e.g., that include the subject nucleic acid compositions, are provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of Application Ser. No.PCT/US00/28477 filed on Oct. 13, 2000; which application is acontinuation-in-part of the following applications: application Ser. No.09/418,529 filed Oct. 14, 1999, now abandoned; application Ser. No.09/418,917 filed Oct. 15, 1999 now abandoned; application Ser. No.09/418,922 filed Oct. 15, 1999 now abandoned; application Ser. No.09/444,338 filed Nov. 19, 1999 now abandoned; application Ser. No.09/444,341 filed Nov. 19, 1999 now abandoned; application Ser. No.09/457,556 filed Dec. 9, 1999 now abandoned; application Ser. No.09/458,477 filed Dec. 9, 1999 now abandoned; application Ser. No.09/458,144 filed Dec. 9, 1999 now abandoned; and application Ser. No.09/457,898 filed Dec. 9, 1999 now abandoned; all of which applicationsclaim priority to application Ser. No. 09/210,330 filed Dec. 11, 1998now abandoned; as well as application Ser. No. 60/211,627 filed on Jun.14, 2000; application Ser. No. 60/211,687 filed on Jun. 14, 2000;application serial No. 60/211,609 filed on Jun. 14, 2000; applicationSer. No. 60/211,626 filed on Jun. 14, 2000; application Ser. No.60/211,880 filed on Jun. 14, 2000; application Ser. No. 60/211,607 filedon Jun. 14, 2000; application Ser. No. 60/211,766 filed on Jun. 14,2000; application Ser. No. 60/211,888 filed on Jun. 14, 2000; andapplication Ser. No. 60/212,070 filed on Jun. 14, 2000; as well asInternational Application Ser. No. PCT/US99/29405 filed Dec. 10, 1999,which application claims priority to application Ser. No. 09/210,330filed Dec. 11, 1998; the disclosures of which application areincorporated in their entirety herein.

INTRODUCTION

1. Field of the Invention

The field of this invention is chromoproteins and fluorescent proteins.

2. Background of the Invention

Labeling is a tool for marking a protein, cell, or organism of interestand plays a prominent role in many biochemistry, molecular biology andmedical diagnostic applications. A variety of different labels have beendeveloped, including radiolabels, chromolabels, fluorescent labels,chemiluminescent labels, etc. However, there is continued interest inthe development of new labels. Of particular interest is the developmentof new protein labels, including chromo- and/or fluorescent proteinlabels.

Relevant Literature

U.S. Patents of interest include: U.S. Pat. Nos. 6,066,476; 6,020,192;5,985,577; 5,976,796; 5,968,750; 5,968,738; 5,958,713; 5,919,445;5,874,304; and 5,491,084. International Patent Publications of interestinclude: WO 00/46233; WO 99/49019; and DE 197 18 640 A. Also of interestare: Anderluh et al., Biochemical and Biophysical ResearchCommunications (1996) 220:437–442; Dove et al., Biological Bulletin(1995) 189:288–297; Fradkov et al., FEBS Lett. (2000) 479(3):127–30;Gurskaya et al., FEBS Left., (2001) 507(1):16–20; Gurskaya et al., BMCBiochem. (2001) 2:6; Lukyanov, K., et al (2000) J Biol Chemistry275(34):25879–25882; Macek et al., Eur. J. Biochem. (1995) 234:329–335;Martynov et al., J Biol Chem. (2001) 276:21012–6; Matz, M. V., et al.(1999) Nature Biotechnol., 17:969–973; Terskikh et al., Science (2000)290:1585–8; Tsien, Annual Rev. of Biochemistry (1998) 67:509–544; Tsien,Nat. Biotech. (1999) 17:956–957; Ward et al., J. Biol. Chem. (1979)254:781–788; Wiedermann et al., Jarhrestagung der Deutschen Gesellschactfur Tropenokologie-gto. Ulm. 17–19 Feb. 02, 1999. Poster P-4.20;Yarbrough et al., Proc Natl Acad Sci U S A (2001) 98:462–7.

SUMMARY OF THE INVENTION

Nucleic acid compositions encoding novel chromo/fluoroproteins andmutants thereof, as well as the proteins encoded the same, are provided.The proteins of interest are proteins that are colored and/orfluorescent, where this feature arises from the interaction of two ormore residues of the protein. The subject proteins are furthercharacterized in that they are either obtained from non-bioluminescentCnidarian, e.g., Anthozoan, species or are obtained from Anthozoannon-Pennatulacean (sea pen) species. Specific proteins of interestinclude proteins obtained from the following specific Anthozoan species:Anemonia majano (NFP-1), Clavularia sp. (NFP-2), Zoanthus sp. (NFP-3 &NFP-4), Discosoma striata (NFP-5), Discosoma sp. “red”(NFP-6), Anemoniasulcata (NFP-7), Discosoma sp “green” (NFP-8), and Discosoma sp.“magenta” (NFP-9). Also of interest are proteins that are substantiallysimilar to, or mutants of, the above specific proteins. Also providedare fragments of the nucleic acids and the peptides encoded thereby, aswell as antibodies to the subject proteins and transgenic cells andorganisms. The subject protein and nucleic acid compositions find use ina variety of different applications. Finally, kits for use in suchapplications, e.g., that include the subject nucleic acid compositions,are provided.

BREIF DESCRIPTION OF THE FIGURES

FIG. 1 provides the nucleotide and amino acid sequence of wild typeamFP486 (NFP-1). (SEQ ID NO:01 & 02)

FIG. 2 provides the nucleotide and amino acid sequence of wild typecFP484 (NFP-2). (SEQ ID NO:03 & 04)

FIG. 3 provides the nucleotide and amino acid sequence of wild typezFP506 (NFP-3). (SEQ ID NO:05 & 06)

FIG. 4 provides the nucleotide and amino acid sequence of wild typezFP538 (NFP-4). (SEQ ID NO:07 & 08)

FIG. 5 provides the nucleotide and amino acid sequence of wild typedsFP483 NFP-5). (SEQ ID NO:09 & 10)

FIG. 6 provides the nucleotide and amino acid sequence of wild typedrFP583 (NFP-6). (SEQ ID NO:11 & 12); as well as the nucleotide andamino acid sequence of an alternative version thereof.

FIG. 7 provides the nucleotide and amino acid sequence of wild typeasFP600 (NFP-7). (SEQ ID NO:13 & 14)

FIG. 8 provides the nucleotide and amino acid sequence of wild typedgFP512 (NFP-8). (SEQ ID NO:15 & 16)

FIG. 9 provides the nucleotide and amino acid sequence of wild typedmFP592 (NFP-9). (SEQ ID NO:17 & 18)

FIG. 10 provides the nucleotide and amino acid sequence of mut32-NA (SEQID NO: 27 & 28)

FIG. 11 provides the nucleotide and amino acid sequence of FP3-NA (SEQID NO:29 & 30)

FIG. 12 provides the amino acid sequence of FP3-yellow (SEQ ID NO:31)

FIG. 13 provides the amino acid sequence of FP3-Yellow bright (SEQ IDNO:32)

FIG. 14 provides the nucleotide and amino acid sequence of NFP4-NA (SEQID NO:33 & 34).

FIG. 15 provides additional sequence information of NFP-6 mutants.

FIG. 16 provides the nucleotide sequence of humanized NFP-6. (SEQ IDNO:35)

FIG. 17 provides the nucleotide sequence of mutant E5-NA. (SEQ ID NO:36)

FIG. 18 provides the nucleotide sequence of mutant E57. (SEQ ID NO:37)

FIG. 19 provides the nucleotide sequence of mutant E57-NA (SEQ ID NO:38)

FIG. 20 provides the nucleotide and amino acid sequence of mutant FP7-NA(SEQ ID NO:39 & 40).

FIG. 21 provides the nucleotide and amino acid sequence of humanizedFP7. (SEQ ID NO:41 & 42).

FIG. 22 provides the nucleotide and amino acid sequence of humanized6/9Q (SEQ ID NO:43 & 44).

DEFINITIONS

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Maniatis, Fritsch & Sambrook,“Molecular Cloning: A Laboratory Manual (1982); “DNA Cloning: APractical Approach,” Volumes I and II (D. N. Glover ed. 1985);“Oligonucleotide Synthesis” (M. J. Gait ed. 1984); “Nucleic AcidHybridization” (B. D. Hames & S. J. Higgins eds. (1985)); “Transcriptionand Translation” (B. D. Hames & S. J. Higgins eds. (1984)); “Animal CellCulture” (R. I. Freshney, ed. (1986)); “Immobilized Cells and Enzymes”(IRL Press, (1986)); B. Perbal, “A Practical Guide To Molecular Cloning”(1984).

A “vector” is a replicon, such as plasmid, phage or cosmid, to whichanother DNA segment may be attached so as to bring about the replicationof the attached segment.

A “DNA molecule” refers to the polymeric form of deoxyribonucleotides(adenine, guanine, thymine, or cytosine) in either single stranded formor a double-stranded helix. This term refers only to the primary andsecondary structure of the molecule, and does not limit it to anyparticular tertiary forms. Thus, this term includes double-stranded DNAfound, inter alia, in linear DNA molecules (e.g., restrictionfragments), viruses, plasmids, and chromosomes.

A DNA “coding sequence” is a DNA sequence which is transcribed andtranslated into a polypeptide in vivo when placed under the control ofappropriate regulatory sequences. The boundaries of the coding sequenceare determined by a start codon at the 5′ (amino) terminus and atranslation stop codon at the 3′ (carboxyl) terminus. A coding sequencecan include, but is not limited to, prokaryotic sequences, cDNA fromeukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian)DNA, and synthetic DNA sequences. A polyadenylation signal andtranscription termination sequence may be located 3′ to the codingsequence.

As used herein, the term “hybridization” refers to the process ofassociation of two nucleic acid strands to form an antiparallel duplexstabilized by means of hydrogen bonding between residues of the oppositenucleic acid strands.

The term “oligonucleotide” refers to a short (under 100 bases in length)nucleic acid molecule.

“DNA regulatory sequences”, as used herein, are transcriptional andtranslational control sequences, such as promoters, enhancers,polyadenylation signals, terminators, and the like, that provide forand/or regulate expression of a coding sequence in a host cell.

A “promoter sequence” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. For purposes of defining the presentinvention, the promoter sequence is bounded at its 3′ terminus by thetranscription initiation site and extends upstream (5′ direction) toinclude the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site, as well asprotein binding domains responsible for the binding of RNA polymerase.Eukaryotic promoters will often, but not always, contain “TATA” boxesand “CAT” boxes. Various promoters, including inducible promoters, maybe used to drive the various vectors of the present invention.

As used herein, the terms “restriction endonucleases” and “restrictionenzymes” refer to bacterial enzymes, each of which cut double-strandedDNA at or near a specific nucleotide sequence.

A cell has been “transformed” or “transfected” by exogenous orheterologous DNA when such DNA has been introduced inside the cell. Thetransforming DNA may or may not be integrated (covalently linked) intothe genome of the cell. In prokaryotes, yeast, and mammalian cells forexample, the transforming DNA may be maintained on an episomal elementsuch as a plasmid. With respect to eukaryotic cells, a stablytransformed cell is one in which the transforming DNA has becomeintegrated into a chromosome so that it is inherited by daughter cellsthrough chromosome replication. This stability is demonstrated by theability of the eukaryotic cell to establish cell lines or clonescomprised of a population of daughter cells containing the transformingDNA. A “clone” is a population of cells derived from a single cell orcommon ancestor by mitosis. A “cell line” is a clone of a primary cellthat is capable of stable growth in vitro for many generations.

A “heterologous” region of the DNA construct is an identifiable segmentof DNA within a larger DNA molecule that is not found in associationwith the larger molecule in nature. Thus, when the heterologous regionencodes a mammalian gene, the gene will usually be flanked by DNA thatdoes not flank the mammalian genomic DNA in the genome of the sourceorganism. In another example, heterologous DNA includes coding sequencein a construct where portions of genes from two different sources havebeen brought together so as to produce a fusion protein product. Allelicvariations or naturally-occurring mutational events do not give rise toa heterologous region of DNA as defined herein.

As used herein, the term “reporter gene” refers to a coding sequenceattached to heterologous promoter or enhancer elements and whose productmay be assayed easily and quantifiably when the construct is introducedinto tissues or cells.

The amino acids described herein are preferred to be in the “L” isomericform. The amino acid sequences are given in one-letter code (A: alanine;C: cysteine; D: aspartic acid; E: glutamic acid; F: phenylalanine; G:glycine; H: histidine; I: isoleucine; K: lysine; L: leucine; M:methionine; N: asparagine; P: proline; Q: glutamine; R: arginine; S:serine; T: threonine; V: valine; W: tryptophan; Y: tyrosine; X: anyresidue). NH₂ refers to the free amino group present at the aminoterminus of a polypeptide. COOH refers to the free carboxy group presentat the carboxy terminus of a polypeptide. In keeping with standardpolypeptide nomenclature, J Biol. Chem., 243 (1969), 3552–59 is used.

The term “immunologically active” defines the capability of the natural,recombinant or synthetic chromo/fluorescent protein, or any oligopeptidethereof, to induce a specific immune response in appropriate animals orcells and to bind with specific antibodies. As used herein, “antigenicamino acid sequence” means an amino acid sequence that, either alone orin association with a carrier molecule, can elicit an antibody responsein a mammal. The term “specific binding,” in the context of antibodybinding to an antigen, is a term well understood in the art and refersto binding of an antibody to the antigen to which the antibody wasraised, but not other, unrelated antigens.

As used herein the term “isolated” is meant to describe apolynucleotide, a polypeptide, an antibody, or a host cell that is in anenvironment different from that in which the polynucleotide, thepolypeptide, the antibody, or the host cell naturally occurs.

Bioluminescence (BL) is defined as emission of light by living organismsthat is well visible in the dark and affects visual behavior of animals(See e.g., Harvey, E. N. (1952). Bioluminescence. New York: AcademicPress; Hastings, J. W. (1995). Bioluminescence. In: Cell Physiology (ed.by N. Speralakis). pp. 651–681. New York: Academic Press.; Wilson, T.and Hastings, J. W. (1998). Bioluminescence. Annu Rev Cell Dev Biol 14,197–230.). Bioluminescence does not include so-called ultra-weak lightemission, which can be detected in virtually all living structures usingsensitive luminometric equipment (Murphy, M. E. and Sies, H.(1990).Visible-range low-level chemiluminescence in biological systems. Meth.Enzymol. 186, 595–610; Radotic, K, Radenovic, C, Jeremic, M. (1998.)Spontaneous ultra-weak bioluminescence in plants: origin, mechanisms andproperties. Gen Physiol Biophys 17, 289–308), and from weak lightemission which most probably does not play any ecological role, such asthe glowing of bamboo growth cone (Totsune, H., Nakano, M., Inaba,H.(1993). Chemiluminescence from bamboo shoot cut. Biochem. Biophys. ResComm. 194, 1025–1029) or emission of light during fertilization ofanimal eggs (Klebanoff, S. J., Froeder, C. A., Eddy, E. M., Shapiro, B.M. (1979). Metabolic similarities between fertilization andphagocytosis. Conservation of peroxidatic mechanism. J. Exp. Med. 149,938–953; Schomer, B. and Epel, D. (1998). Redox changes duringfertilization and maturation of marine invertebrate eggs. Dev Biol 203,1–11).

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Nucleic acid compositions encoding novel chromo/fluoroproteins andmutants thereof, as well as the proteins encoded the same, are provided.The proteins of interest are proteins that are colored and/orfluorescent, where this feature arises from the interaction of two ormore residues of the protein. The subject proteins are furthercharacterized in that they are either obtained from non-bioluminescentCnidarian, e.g., Anthozoan, species or are obtained fromnon-Pennatulacean (sea pen) Anthozoan species. Specific proteins ofinterest include proteins obtained from the following specific Anthozoanspecies: Anemonia majano (NFP-1), Clavularia sp. (NFP-2), Zoanthus sp.(NFP-3 & NFP-4), Discosoma striata (NFP-5), Discosoma sp. “red” (NFP-6),Anemonia sulcata (NFP-7), Discosoma sp “green” (NFP-8), and Discosomasp. “magenta” (NFP-9). Also of interest are proteins that aresubstantially similar to, or mutants of, the above specific proteins.Also provided are fragments of the nucleic acids and the peptidesencoded thereby, as well as antibodies to the subject proteins, andtransgenic cells and organisms that include the subject nucleicacid/protein compositions. The subject protein and nucleic acidcompositions find use in a variety of different applications. Finally,kits for use in such applications, e.g., that include the subjectnucleic acid compositions, are provided.

Before the subject invention is described further, it is to beunderstood that the invention is not limited to the particularembodiments of the invention described below, as variations of theparticular embodiments may be made and still fall within the scope ofthe appended claims. It is also to be understood that the terminologyemployed is for the purpose of describing particular embodiments, and isnot intended to be limiting. Instead, the scope of the present inventionwill be established by the appended claims.

In this specification and the appended claims, the singular forms “a,”“an” and “the” include plural reference unless the context clearlydictates otherwise. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood to one of ordinary skill in the art to which this inventionbelongs.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range, and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention belongs. Although any methods, devicesand materials similar or equivalent to those described herein can beused in the practice or testing of the invention, the preferred methods,devices and materials are now described.

All publications mentioned herein are incorporated herein by referencefor the purpose of describing and disclosing the cell lines, vectors,methodologies and other invention components that are described in thepublications which might be used in connection with the presentlydescribed invention.

In further describing the subject invention, the subject nucleic acidcompositions will be described first, followed by a discussion of thesubject protein compositions, antibody compositions and transgeniccells/organisms. Next a review of representative methods in which thesubject proteins find use is provided.

Nucleic Acid Compositions

As summarized above, the subject invention provides nucleic acidcompositions encoding chromo- and fluoroproteins and mutants thereof, aswell as fragments and homologues of these proteins. By chromo and/orfluorescent protein is meant a protein that is colored, i.e., ispigmented, where the protein may or may not be fluorescent, e.g., it mayexhibit low, medium or high fluorescence upon irradiation with light ofan excitation wavelength. In any event, the subject proteins of interestare those in which the colored characteristic, i.e., the chromo and/orfluorescent characteristic, is one that arises from the interaction oftwo or more residues of the protein, and not from a single residue, morespecifically a single side chain of a single residue, of the protein. Assuch, fluorescent proteins of the subject invention do not includeproteins that exhibit fluorescence only from residues that act bythemselves as intrinsic fluors, i.e., tryptophan, tyrosine andphenylalanine. As such, the fluorescent proteins of the subjectinvention are fluorescent proteins whose fluorescence arises from somestructure in the protein that is other than the above specified singleresidues, e.g., it arises from an interaction of two or more residues.

By nucleic acid composition is meant a composition comprising a sequenceof DNA having an open reading frame that encodes a chromo/fluoropolypeptide of the subject invention, i.e., a chromo/fluoroprotein gene,and is capable, under appropriate conditions, of being expressed as achromo/fluoro protein according to the subject invention. Alsoencompassed in this term are nucleic acids that are homologous,substantially similar or identical to the nucleic acids of the presentinvention. Thus, the subject invention provides genes and codingsequences thereof encoding the proteins of the subject invention, aswell as homologs thereof. The subject nucleic acids are present in otherthan their natural environment, e.g., they are isolated, present inenriched amounts, etc., from their naturally occurring environment,e.g., the organism from which they are obtained.

The nucleic acids are further characterized in that they encode proteinsthat are either from: (1) non-bioluminescent species, oftennon-bioluminescent Cnidarian species, e.g., non-bioluminescent Anthozoanspecies; or (2) from Anthozoan species that are not Pennatulaceanspecies, i.e., that are not sea pens. As such, the nucleic acids mayencode proteins from bioluminescent Anthozoan species, so long as thesespecies are not Pennatulacean species, e.g., that are not Renillan orPtilosarcan species. Specific nucleic acid compositions of interest arethose that encode proteins (and mutants thereof) from the followingspecific Anthozoan species: Anemonia majano, Clavularia sp., Zoanthussp., Discosoma striata, Discosoma sp. “red”, Anemonia sulcata, Discosomasp “green”, and Discosoma sp. “magenta.” Each of these particular typesof nucleic acid compositions of interest is now discussed in greaterdetail individually.

Anemonia majano (NFP-1; AmCyan; RCFP-1)

In these embodiments, the nucleic acid compositions are found in,derived from, or are mutants, homologues of, a nucleic acid found in anorganism from the Anthozoan Sub-class Zoantharia, often OrderActiniaria, more often Sub-order Endomyaria, usually Family Actiniidae,and more usually Genus Anemonia, where in many embodiments, the organismis Anemonia majano, where the specific wild type protein of interest isamFP486 (i.e., NFP-1; RCFP-1). The wild type cDNA coding sequence foramFP486 is provided in SEQ ID NO: 01. In addition to nucleic acidsencoding the wild type protein and fragments thereof, also of interestare nucleic acids that encode homologues and mutants of the wild typeprotein. Specific mutants of interest include, but are not limited to:Mut15, Mut32, and FP1-NA (a non-aggregating mutant), where thesespecific mutants are further described in the experimental section,infra.

Clavularia sp. (NFP-2; RCPF-2)

In these embodiments, the nucleic acid compositions are found in,derived from, or are mutants or homologues of, nucleic acids found inAnthozoan organisms from Sub-class Alcyonaria, often Order Stolonifera,and more often the Family Clavulariidae, where the organism is usuallyfrom the Genus Clavularia, and in certain embodiments, the organism isClavularia sp., where the specific wild type fluorescent protein ofinterest is cFP484 (i.e., NFP-2; RCFP-2). The wild type cDNA codingsequence for cFP484 is provided in SEQ ID NO: 03. In addition to nucleicacids encoding the wild type sequence and fragments thereof, also ofinterest are nucleic acids that encode homologues and mutants of thewild type protein. Specific mutants of interest include, but are notlimited to: Δ19 cFP484 and Δ38 cFP484, where these specific mutants arefurther described in the experimental section, infra.

Zoanthus sp. I (NFP-3; ZsGreen; RCFP-3)

In these embodiments, the nucleic acid compositions are found in,derived from, or are mutants or homologues of, nucleic acids found inAnthozoan organisms from Sub-class Zoantharia, often Order Zoanthidea,more often Sub-order Brachycnemia, usually Family Zoanthidae, and moreusually Genus Zoanthus, where in certain embodiments, the organism isZoanthus sp., where the specific wild type fluorescent protein ofinterest is zFP506 (i.e., NFP-3; RCFP-3). The wild type cDNA codingsequence for zFP506 is provided in SEQ ID NO: 05. In addition to nucleicacids encoding the wild type sequence and fragments thereof, also ofinterest are nucleic acids that encode homologues and mutants of thewild type protein. Specific mutants of interest include, but are notlimited to: N66M; NFP-3NA (a non-aggregating mutant); yellow; yellowbright, etc., where these specific mutants are further described in theexperimental section, infra.

Zoanthus sp. II (NFP-4; ZsYellow; RCFP-4)

In these embodiments, the nucleic acid compositions are found in,derived from, or are mutants or homologues of, nucleic acids found inAnthozoan organisms from Sub-class Zoantharia, often Order Zoanthidea,more often Sub-order Brachycnemia, usually Family Zoanthidae, and moreusually Genus Zoanthus, where in certain embodiments, the organism isZoanthus sp., where the specific wild type fluorescent protein ofinterest is zFP538 (NFP-4; RCFP-4). The wild type cDNA coding sequencefor zFP538 is provided in SEQ ID NO: 07. In addition to nucleic acidsencoding the wild type sequence and fragments thereof, also of interestare nucleic acids that encode homologues and mutants of the wild typeprotein. Specific mutants of interest include, but are not limited to:M129V; FP4-NA (a non-aggregating mutant); green; etc., where thesespecific mutants are further described in the experimental section,infra.

Discosoma striata (NFP-5; RCFP-5)

In these embodiments, the nucleic acid compositions are found in,derived from, or are mutants or homologues of, nucleic acids found inAnthozoan organisms from Sub-class Zoantharia, often OrderCorallimopharia, more often Family Discosomatidae, and usually GenusDiscosoma, where in certain embodiments, the organism is Discosomastriata, where the specific wild type fluorescent protein of interest isdsFP483 (NFP-5; RCFP-5). The wild type cDNA coding sequence for dsFP483is provided in SEQ ID NO: 09. In addition to nucleic acids encoding thewild type sequence and fragments thereof, also of interest are nucleicacids that encode homologues and mutants of the wild type protein.

Discosoma sp. “red” (NFP-6; RCFP-6; DsRed; DsRed2)

In these embodiments, the nucleic acid compositions are found in,derived from, or are mutants or homologues of, nucleic acids found inAnthozoan organisms from Sub-class Zoantharia, often OrderCorallimopharia, more often Family Discosomatidae, and usually GenusDiscosoma, where in certain embodiments, the organism is Discosoma sp.“red”., where the specific wild type fluorescent protein of interest isdrFP583 (NFP-6; RCFP-6). The wild type cDNA coding sequence for drFP583is provided in SEQ ID NO: 11. In addition to nucleic acids encoding thewild type sequence and fragments thereof, also of interest are nucleicacids that encode homologues and mutants of the wild type protein.Specific mutants of interest include, but are not limited to: E5, E5-NA(a non-aggregating mutant); E8, E5 up, E5 down, E57, FP6-NA (anon-aggregating mutant), AG4, AG45, E83, 6/9 Q, 6/9 Q-NA, 6/92G; etc.,where these specific mutants are further described in the experimentalsection, infra.

Anemonia sulcata (NFP-7; AsRed; RCFP-7)

In these embodiments, the nucleic acid compositions are found in,derived from, or are mutants or homologues of, nucleic acids found inAnthozoan organisms from Sub-class Zoantharia, often Order Actiniaria,more often Sub-Order Endomyaria, usually Family Actiniidae, and moreusually Genus Anemonia, e.g., where in certain embodiments the organismis Anemonia sulcata, where the specific wild type fluorescent protein ofinterest is asFP600 (NFP-7; RCFP-7). The wild type cDNA coding sequencefor asFP600 is provided in SEQ ID NO:14. In addition to nucleic acidsencoding the wild type sequence and fragments thereof, also of interestare nucleic acids that encode homologues and mutants of the wild typeprotein. Specific mutants of interest include, but are not limited to:Mut1; Mut35–5/Mut1; FP7-NA (a non-aggregating mutant), etc., where thesespecific mutants are further described in the experimental section,infra.

Discosoma sp “green” (NFP-8; RCFP-8)

In these embodiments, the nucleic acid compositions are found in,derived from, or are mutants or homologues of, nucleic acids found inAnthozoan organisms from Sub-class Zoantharia, often OrderCorallimopharia, more often Family Discosomatidae, and usually GenusDiscosoma, where in certain embodiments, the organism is Discosoma sp.“green”., where the specific wild type fluorescent protein of interestis dgFP512 (NFP-8; RCFP-8). The wild type cDNA coding sequence fordgFP512 is provided in SEQ ID NO: 15. In addition to nucleic acidsencoding the wild type sequence and fragments thereof, also of interestare nucleic acids that encode homologues and mutants of the wild typeprotein.

Discosoma sp. “magenta” (NFP-9; RCFP-9)

In these embodiments, the nucleic acid compositions are found in,derived from, or are mutants or homologues of, nucleic acids found inAnthozoan organisms from Sub-class Zoantharia, often OrderCorallimopharia, more often Family Discosomatidae, and usually GenusDiscosomam where in certain embodiments, the organism is Discosoma sp.“magenta”., where the specific wild type fluorescent protein of interestis dmFP592 (NFP-9; RCFP-9). The wild type cDNA coding sequence fordmFP592 is provided in SEQ ID NO: 17. In addition to nucleic acidsencoding the wild type sequence and fragments thereof, also of interestare nucleic acids that encode homologues and mutants of the wild typeprotein.

In addition to the above described specific nucleic acid compositions,also of interest are homologues of the above sequences. With respect tohomologues of the subject nucleic acids, the source of homologous genesmay be any species of plant or animal or the sequence may be wholly orpartially synthetic. In certain embodiments, sequence similarity betweenhomologues is at least about 20%, sometimes at least about 25%, and maybe 30%, 35%, 40%, 50%, 60%, 70% or higher, including 75%, 80%, 85%, 90%and 95% or higher. Sequence similarity is calculated based on areference sequence, which may be a subset of a larger sequence, such asa conserved motif, coding region, flanking region, etc. A referencesequence will usually be at least about 18 nt long, more usually atleast about 30 nt long, and may extend to the complete sequence that isbeing compared. Algorithms for sequence analysis are known in the art,such as BLAST, described in Altschul et al. (1990), J. Mol. Biol.215:403–10 (using default settings, i.e. parameters w=4 and T=17). Thesequences provided herein are essential for recognizing related andhomologous nucleic acids in database searches. Of particular interest incertain embodiments are nucleic acids of substantially the same lengthas the nucleic acid identified as SEQ ID NOS: 01, 03, 05, 07, 09, 11,13, 15, or 17, where by substantially the same length is meant that anydifference in length does not exceed about 20 number %, usually does notexceed about 10 number % and more usually does not exceed about 5 number%; and have sequence identity to any of these sequences of at leastabout 90%, usually at least about 95% and more usually at least about99% over the entire length of the nucleic acid. In many embodiments, thenucleic acids have a sequence that is substantially similar (i.e. thesame as) or identical to the sequences of SEQ ID NOS: 01, 03, 05, 07,09, 11, 13, 15, or 17. By substantially similar is meant that sequenceidentity will generally be at least about 60%, usually at least about75% and often at least about 80, 85, 90, or even 95%.

Also provided are nucleic acids that encode the proteins encoded by theabove described nucleic acids, but differ in sequence from the abovedescribed nucleic acids due to the degeneracy of the genetic code.

Also provided are nucleic acids that hybridize to the above describednucleic acid under stringent conditions. An example of stringenthybridization conditions is hybridization at 50° C. or higher and0.1×SSC (15 mM sodium chloride/1.5 mM sodium citrate). Another exampleof stringent hybridization conditions is overnight incubation at 42° C.in a solution: 50% formamide, 5×SSC (150 mM NaCl, 15 mM trisodiumcitrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10%dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA,followed by washing the filters in 0.1×SSC at about 65° C. Stringenthybridization conditions are hybridization conditions that are at leastas stringent as the above representative conditions, where conditionsare considered to be at least as stringent if they are at least about80% as stringent, typically at least about 90% as stringent as the abovespecific stringent conditions. Other stringent hybridization conditionsare known in the art and may also be employed to identify nucleic acidsof this particular embodiment of the invention.

Nucleic acids encoding mutants of the proteins of the invention are alsoprovided.

Mutant nucleic acids can be generated by random mutagenesis or targetedmutagenesis, using well-known techniques which are routine in the art.In some embodiments, chromo- or fluorescent proteins encoded by nucleicacids encoding homologues or mutants have the same fluorescentproperties as the wild-type fluorescent protein. In other embodiments,homologue or mutant nucleic acids encode chromo- or fluorescent proteinswith altered spectral properties, as described in more detail herein.

One category of mutant that is of particular interest is thenon-aggregating mutant. In many embodiments, the non-aggregating mutantdiffers from the wild type sequence by a mutation in the N-terminus thatmodulates the charges appearing on side groups of the N-terminusresidues, e.g., to reverse or neutralize the charge, in a mannersufficient to produce a non-aggregating mutant of the naturallyoccurring protein or mutant, where a particular protein is considered tobe non-aggregating if it is determined be non-aggregating using theassay reported in U.S. Patent Application Ser. No. 60/270,983, thedisclosure of which is herein incorporated by reference. Morespecifically, basic residues located near the N-termini of the proteinsare substituted, e.g., Lys and Arg residues close to the N-terminus aresubstituted with negatively charged or neutral residues. Specificnon-aggregating mutants of interest include, but are not limited to:FP1-NA; FP3-NA; FP4-NA; FP6-NA; E5-NA; 6/9Q-NA; 7A-NA; and the like,where these particular non-aggregating mutants are further describedinfra.

Another category of mutant of particular interest is the modulatedoligomerization mutant. A mutant is considered to be a modulatedoligomerization mutant if its oligomerization properties are differentas compared to the wild type protein. For example, if a particularmutant oligomerizes to a greater or lesser extent than the wild type, itis considered to be an oligomerization mutant. Of particular interestare oligomerization mutants that do not oligomerize, i.e., are monomersunder physiological (e.g., intracellular) conditions, or oligomerize toa lesser extent that the wild type, e.g., are dimers or trimers underintracellular conditions.

Nucleic acids of the subject invention may be cDNA or genomic DNA or afragment thereof. In certain embodiments, the nucleic acids of thesubject invention include one or more of the open reading framesencoding specific fluorescent proteins and polypeptides, and introns, aswell as adjacent 5′ and 3′ non-coding nucleotide sequences involved inthe regulation of expression, up to about 20 kb beyond the codingregion, but possibly further in either direction. The subject nucleicacids may be introduced into an appropriate vector for extrachromosomalmaintenance or for integration into a host genome, as described ingreater detail below.

The term “cDNA” as used herein is intended to include all nucleic acidsthat share the arrangement of sequence elements found in native maturemRNA species, where sequence elements are exons and 5′ and 3′ non-codingregions. Normally mRNA species have contiguous exons, with theintervening introns, when present, being removed by nuclear RNAsplicing, to create a continuous open reading frame encoding theprotein.

A genomic sequence of interest comprises the nucleic acid presentbetween the initiation codon and the stop codon, as defined in thelisted sequences, including all of the introns that are normally presentin a native chromosome. It may further include 5′ and 3′ un-translatedregions found in the mature mRNA. It may further include specifictranscriptional and translational regulatory sequences, such aspromoters, enhancers, etc., including about 1 kb, but possibly more, offlanking genomic DNA at either the 5′ or 3′ end of the transcribedregion. The genomic DNA may be isolated as a fragment of 100 kbp orsmaller; and substantially free of flanking chromosomal sequence. Thegenomic DNA flanking the coding region, either 3′ or 5′, or internalregulatory sequences as sometimes found in introns, contains sequencesrequired for proper tissue and stage specific expression.

The nucleic acid compositions of the subject invention may encode all ora part of the subject proteins. Double or single stranded fragments maybe obtained from the DNA sequence by chemically synthesizingoligonucleotides in accordance with conventional methods, by restrictionenzyme digestion, by PCR amplification, etc. For the most part, DNAfragments will be of at least about 15 nt, usually at least about 18 ntor about 25 nt, and may be at least about 50 nt. In some embodiments,the subject nucleic acid molecules may be about 100 nt, about 200 nt,about 300 nt, about 400 nt, about 500 nt, about 600 nt, about 700 nt, orabout 720 nt in length. The subject nucleic acids may encode fragmentsof the subject proteins or the full-length proteins, e.g., the subjectnucleic acids may encode polypeptides of about 25 aa, about 50 aa, about75 aa, about 100 aa, about 125 aa, about 150 aa, about 200 aa, about 210aa, about 220 aa, about 230 aa, or about 240 aa, up to the entireprotein.

The subject nucleic acids are isolated and obtained in substantialpurity, generally as other than an intact chromosome. Usually, the DNAwill be obtained substantially free of other nucleic acid sequences thatdo not include a nucleic acid of the subject invention or fragmentthereof, generally being at least about 50%, usually at least about 90%pure and are typically “recombinant”, i.e. flanked by one or morenucleotides with which it is not normally associated on a naturallyoccurring chromosome.

The subject polynucleotides (e.g., a polynucleotide having a sequence ofSEQ ID NOS: 01 to 17 etc.), the corresponding cDNA, the full-length geneand constructs of the subject polynucleotides are provided. Thesemolecules can be generated synthetically by a number of differentprotocols known to those of skill in the art. Appropriate polynucleotideconstructs are purified using standard recombinant DNA techniques asdescribed in, for example, Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd Ed., (1989) Cold Spring Harbor Press, Cold SpringHarbor, N.Y., and under current regulations described in United StatesDept. of HHS, National Institute of Health (NIH) Guidelines forRecombinant DNA Research.

Also provided are nucleic acids that encode fusion proteins of thesubject proteins, or fragments thereof, which are fused to a secondprotein, e.g., a degradation sequence, a signal peptide, etc. Fusionproteins may comprise a subject polypeptide, or fragment thereof, and anon-Anthozoan polypeptide (“the fusion partner”) fused in-frame at theN-terminus and/or C-terminus of the subject polypeptide. Fusion partnersinclude, but are not limited to, polypeptides that can bind antibodyspecific to the fusion partner (e.g., epitope tags); antibodies orbinding fragments thereof; polypeptides that provide a catalyticfunction or induce a cellular response; ligands or receptors or mimeticsthereof; and the like. In such fusion proteins, the fusion partner isgenerally not naturally associated with the subject Anthozoan portion ofthe fusion protein, and is typically not an Anthozoan protein orderivative/fragment thereof, i.e., it is not found in Anthozoan species.

Also provided are constructs comprising the subject nucleic acidsinserted into a vector, where such constructs may be used for a numberof different applications, including propagation, protein production,etc. Viral and non-viral vectors may be prepared and used, includingplasmids. The choice of vector will depend on the type of cell in whichpropagation is desired and the purpose of propagation. Certain vectorsare useful for amplifying and making large amounts of the desired DNAsequence. Other vectors are suitable for expression in cells in culture.Still other vectors are suitable for transfer and expression in cells ina whole animal or person. The choice of appropriate vector is wellwithin the skill of the art. Many such vectors are availablecommercially. To prepare the constructs, the partial or full-lengthpolynucleotide is inserted into a vector typically by means of DNAligase attachment to a cleaved restriction enzyme site in the vector.Alternatively, the desired nucleotide sequence can be inserted byhomologous recombination in vivo. Typically this is accomplished byattaching regions of homology to the vector on the flanks of the desirednucleotide sequence. Regions of homology are added by ligation ofoligonucleotides, or by polymerase chain reaction using primerscomprising both the region of homology and a portion of the desirednucleotide sequence, for example.

Also provided are expression cassettes or systems that find use in,among other applications, the synthesis of the subject proteins. Forexpression, the gene product encoded by a polynucleotide of theinvention is expressed in any convenient expression system, including,for example, bacterial, yeast, insect, amphibian and mammalian systems.Suitable vectors and host cells are described in U.S. Pat. No.5,654,173. In the expression vector, a subject polynucleotide, e.g., asset forth in SEQ ID NOS:01; 03; 05; 07; 09; 11; 13; 15 or 17, is linkedto a regulatory sequence as appropriate to obtain the desired expressionproperties. These regulatory sequences can include promoters (attachedeither at the 5′ end of the sense strand or at the 3′ end of theantisense strand), enhancers, terminators, operators, repressors, andinducers. The promoters can be regulated or constitutive. In somesituations it may be desirable to use conditionally active promoters,such as tissue-specific or developmental stage-specific promoters. Theseare linked to the desired nucleotide sequence using the techniquesdescribed above for linkage to vectors. Any techniques known in the artcan be used. In other words, the expression vector will provide atranscriptional and translational initiation region, which may beinducible or constitutive, where the coding region is operably linkedunder the transcriptional control of the transcriptional initiationregion, and a transcriptional and translational termination region.These control regions may be native to the subject species from whichthe subject nucleic acid is obtained, or may be derived from exogenoussources.

Expression vectors generally have convenient restriction sites locatednear the promoter sequence to provide for the insertion of nucleic acidsequences encoding heterologous proteins. A selectable marker operativein the expression host may be present. Expression vectors may be usedfor, among other things, the production of fusion proteins, as describedabove.

Expression cassettes may be prepared comprising a transcriptioninitiation region, the gene or fragment thereof, and a transcriptionaltermination region. Of particular interest is the use of sequences thatallow for the expression of functional epitopes or domains, usually atleast about 8 amino acids in length, more usually at least about 15amino acids in length, to about 25 amino acids, and up to the completeopen reading frame of the gene. After introduction of the DNA, the cellscontaining the construct may be selected by means of a selectablemarker, the cells expanded and then used for expression.

The above described expression systems may be employed with prokaryotesor eukaryotes in accordance with conventional ways, depending upon thepurpose for expression. For large scale production of the protein, aunicellular organism, such as E. coli, B. subtilis, S. cerevisiae,insect cells in combination with baculovirus vectors, or cells of ahigher organism such as vertebrates, e.g. COS 7 cells, HEK 293, CHO,Xenopus Oocytes, etc., may be used as the expression host cells. In somesituations, it is desirable to express the gene in eukaryotic cells,where the expressed protein will benefit from native folding andpost-translational modifications. Small peptides can also be synthesizedin the laboratory. Polypeptides that are subsets of the complete proteinsequence may be used to identify and investigate parts of the proteinimportant for function.

Specific expression systems of interest include bacterial, yeast, insectcell and mammalian cell derived expression systems. Representativesystems from each of these categories is are provided below:

Bacteria. Expression systems in bacteria include those described inChang et al., Nature (1978) 275:615; Goeddel et al., Nature (1979)281:544; Goeddel et al., Nucleic Acids Res. (1980) 8:4057; EP 0 036,776;U.S. Pat. No. 4,551,433; DeBoer et al., Proc. Natl. Acad. Sci. (USA)(1983) 80:21–25; and Siebenlist et al., Cell (1980) 20:269.

Yeast. Expression systems in yeast include those described in Hinnen etal., Proc. Natl. Acad. Sci. (USA) (1978) 75:1929; Ito et al., J.Bacteriol. (1983) 153:163; Kurtz et al., Mol. Cell. Biol. (1986) 6:142;Kunze et al., J. Basic Microbiol. (1985) 25:141; Gleeson et al., J. Gen.Microbiol. (1986) 132:3459; Roggenkamp et al., Mol. Gen. Genet. (1986)202:302; Das et al., J. Bacteriol. (1984) 158:1165; De Louvencourt etal., J. Bacteriol. (1983) 154:737; Van den Berg et al., Bio/Technology(1990) 8:135; Kunze et al., J. Basic Microbiol. (1985) 25:141; Cregg etal., Mol. Cell. Biol. (1985) 5:3376; U.S. Pat. Nos. 4,837,148 and4,929,555; Beach and Nurse, Nature (1981) 300:706; Davidow et al., Curr.Genet. (1985) 10:380; Gaillardin et al., Curr. Genet. (1985) 10:49;Ballance et al., Biochem. Biophys. Res. Commun. (1983) 112:284–289;Tilburn et al., Gene (1983) 26:205–221; Yelton et al., Proc. Natl. Acad.Sci. (USA) (1984) 81:1470–1474; Kelly and Hynes, EMBO J. (1985)4:475479; EP 0 244,234; and WO 91/00357.

Insect Cells. Expression of heterologous genes in insects isaccomplished as described in U.S. Pat. No. 4,745,051; Friesen et al.,“The Regulation of Baculovirus Gene Expression”, in: The MolecularBiology Of Baculoviruses (1986) (W. Doerfler, ed.); EP 0 127,839; EP 0155,476; and Vlak et al., J. Gen. Virol. (1988) 69:765–776; Miller etal., Ann. Rev. Microbiol. (1988) 42:177; Carbonell et al., Gene (1988)73:409; Maeda et al., Nature (1985) 315:592–594; Lebacq-Verheyden etal., Mol. Cell. Biol. (1988) 8:3129; Smith et al., Proc. Natl. Acad.Sci. (USA) (1985) 82:8844; Miyajima et al., Gene (1987) 58:273; andMartin et al., DNA (1988) 7:99. Numerous baculoviral strains andvariants and corresponding permissive insect host cells from hosts aredescribed in Luckow et al., Bio/Technology (1988) 6:47–55, Miller etal., Generic Engineering (1986) 8:277–279, and Maeda et al., Nature(1985) 315:592–594.

Mammalian Cells. Mammalian expression is accomplished as described inDijkema et al., EMBO J. (1985) 4:761, Gorman et al., Proc. Natl. Acad.Sci. (USA) (1982) 79:6777, Boshart et al., Cell (1985) 41:521 and U.S.Pat. No. 4,399,216. Other features of mammalian expression arefacilitated as described in Ham and Wallace, Meth. Enz. (1979) 58:44,Barnes and Sato, Anal. Biochem. (1980) 102:255, U.S. Pat. Nos.4,767,704, 4,657,866, 4,927,762, 4,560,655, WO 90/103430, WO 87/00195,and U.S. RE Pat. No. 30,985.

When any of the above host cells, or other appropriate host cells ororganisms, are used to replicate and/or express the polynucleotides ornucleic acids of the invention, the resulting replicated nucleic acid,RNA, expressed protein or polypeptide, is within the scope of theinvention as a product of the host cell or organism. The product isrecovered by any appropriate means known in the art.

Once the gene corresponding to a selected polynucleotide is identified,its expression can be regulated in the cell to which the gene is native.For example, an endogenous gene of a cell can be regulated by anexogenous regulatory sequence inserted into the genome of the cell atlocation sufficient to at least enhance expressed of the gene in thecell. The regulatory sequence may be designed to integrate into thegenome via homologous recombination, as disclosed in U.S. Pat. Nos.5,641,670 and 5,733,761, the disclosures of which are hereinincorporated by reference, or may be designed to integrate into thegenome via non-homologous recombination, as described in WO 99/15650,the disclosure of which is herein incorporated by reference. As such,also encompassed in the subject invention is the production of thesubject proteins without manipulation of the encoding nucleic aciditself, but instead through integration of a regulatory sequence intothe genome of cell that already includes a gene encoding the desiredprotein, as described in the above incorporated patent documents.

Also provided are homologs of the subject nucleic acids. Homologs areidentified by any of a number of methods. A fragment of the providedcDNA may be used as a hybridization probe against a cDNA library fromthe target organism of interest, where low stringency conditions areused. The probe may be a large fragment, or one or more short degenerateprimers. Nucleic acids having sequence similarity are detected byhybridization under low stringency conditions, for example, at 50° C.and 6×SSC (0.9 M sodium chloride/0.09 M sodium citrate) and remain boundwhen subjected to washing at 55° C. in 1×SSC (0.15 M sodiumchloride/0.015 M sodium citrate). Sequence identity may be determined byhybridization under stringent conditions, for example, at 50° C. orhigher and 0.1×SSC (15 mM sodium chloride/1.5 mM sodium citrate).Nucleic acids having a region of substantial identity to the providedsequences, e.g. allelic variants, genetically altered versions of thegene, etc., bind to the provided sequences under stringent hybridizationconditions. By using probes, particularly labeled probes of DNAsequences, one can isolate homologous or related genes.

Also of interest are promoter elements of the subject genomic sequences,where the sequence of the 5′ flanking region may be utilized forpromoter elements, including enhancer binding sites, e.g., that providefor regulation of expression in cells/tissues where the subject proteinsgene are expressed.

Also provided are small DNA fragments of the subject nucleic acids,which fragments are useful as primers for PCR, hybridization screeningprobes, etc. Larger DNA fragments, i.e., greater than 100 nt are usefulfor production of the encoded polypeptide, as described in the previoussection. For use in geometric amplification reactions, such as geometricPCR, a pair of primers will be used. The exact composition of the primersequences is not critical to the invention, but for most applicationsthe primers will hybridize to the subject sequence under stringentconditions, as known in the art. It is preferable to choose a pair ofprimers that will generate an amplification product of at least about 50nt, preferably at least about 100 nt. Algorithms for the selection ofprimer sequences are generally known, and are available in commercialsoftware packages. Amplification primers hybridize to complementarystrands of DNA, and will prime towards each other.

The DNA may also be used to identify expression of the gene in abiological specimen. The manner in which one probes cells for thepresence of particular nucleotide sequences, as genomic DNA or RNA, iswell established in the literature. Briefly, DNA or mRNA is isolatedfrom a cell sample. The mRNA may be amplified by RT-PCR, using reversetranscriptase to form a complementary DNA strand, followed by polymerasechain reaction amplification using primers specific for the subject DNAsequences. Alternatively, the mRNA sample is separated by gelelectrophoresis, transferred to a suitable support, e.g. nitrocellulose,nylon, etc., and then probed with a fragment of the subject DNA as aprobe. Other techniques, such as oligonucleotide ligation assays, insitu hybridizations, and hybridization to DNA probes arrayed on a solidchip may also find use. Detection of mRNA hybridizing to the subjectsequence is indicative of Anthozoan protein gene expression in thesample.

The subject nucleic acids, including flanking promoter regions andcoding regions, may be mutated in various ways known in the art togenerate targeted changes in promoter strength, sequence of the encodedprotein, properties of the encoded protein, including fluorescentproperties of the encoded protein, etc. The DNA sequence or proteinproduct of such a mutation will usually be substantially similar to thesequences provided herein, e.g. will differ by at least one nucleotideor amino acid, respectively, and may differ by at least two but not morethan about ten nucleotides or amino acids. The sequence changes may besubstitutions, insertions, deletions, or a combination thereof.Deletions may further include larger changes, such as deletions of adomain or exon, e.g. of stretches of 10, 20, 50, 75, 100, 150 or more aaresidues. Techniques for in vitro mutagenesis of cloned genes are known.Examples of protocols for site specific mutagenesis may be found inGustin et al. (1993), Biotechniques 14:22; Barany (1985), Gene37:111–23; Colicelli et al. (1985), Mol. Gen. Genet. 199:537–9; andPrentki et al. (1984), Gene 29:303–13. Methods for site specificmutagenesis can be found in Sambrook et al., Molecular Cloning: ALaboratory Manual, CSH Press 1989, pp. 15.3–15.108; Weiner et al.(1993), Gene 126:35–41; Sayers et al. (1992), Biotechniques 13:592–6;Jones and Winistorfer (1992), Biotechniques 12:528–30; Barton et al.(1990), Nucleic Acids Res 18:7349–55; Marotti and Tomich (1989), GeneAnal. Tech. 6:67–70; and Zhu (1989), Anal Biochem 177:120–4. Suchmutated nucleic acid derivatives may be used to study structure-functionrelationships of a particular chromo/fluorescent protein, or to alterproperties of the protein that affect its function or regulation.

Of particular interest in many embodiments is the following specificmutation protocol, which protocol finds use in mutating chromoproteins(e.g., colored proteins that have little if any fluorescence) intofluorescent mutants. In this protocol, the sequence of the candidateprotein is aligned with the amino acid sequence of Aequorea victoriawild type GFP, according to the protocol reported in Matz et al.,“Fluorescent proteins from non-bioluminescent Anthozoa species,” NatureBiotechnology (October 1999) 17: 969–973. Residue 148 of the alignedchromoprotein is identified and then changed to Ser, e.g., by sitedirected mutagenesis, which results in the production of a fluorescentmutant of the wild type chromoprotein. See e.g., NFP-7 described below,which wild type protein is a chromoprotein that is mutated into afluorescent protein by substitution of Ser for the native Ala residue atposition 148.

Also of interest are humanized versions of the subject nucleic acids. Asused herein, the term “humanized” refers to changes made to the anucleic acid sequence to optimize the codons for expression of theprotein in human cells (Yang et al., Nucleic Acids Research 24 (1996),4592–4593). See also U.S. Pat. No. 5,795,737 which describeshumanization of proteins, the disclosure of which is herein incorporatedby reference.

Protein/Polypeptide Compositions

Also provided by the subject invention are chromo- and/or fluorescentproteins and mutants thereof, as well as polypeptide compositionsrelated thereto. As the subject proteins are chromoproteins, they arecolored proteins, which may be fluorescent, low or non-fluorescent. Asused herein, the terms chromoprotein and fluorescent protein do notinclude luciferases, such as Renilla luciferase, and refer to anyprotein that is pigmented or colored and/or fluoresces when irradiatedwith light, e.g., white light or light of a specific wavelength (ornarrow band of wavelengths such as an excitation wavelength). The termpolypeptide composition as used herein refers to both the full-lengthprotein, as well as portions or fragments thereof. Also included in thisterm are variations of the naturally occurring protein, where suchvariations are homologous or substantially similar to the naturallyoccurring protein, and mutants of the naturally occurring proteins, asdescribed in greater detail below. The subject polypeptides are presentin other than their natural environment.

In many embodiments, the subject proteins have an absorbance maximumranging from about 300 to 700, usually from about 350 to 650 and moreusually from about 400 to 600 nm. Where the subject proteins arefluorescent proteins, by which is meant that they can be excited at onewavelength of light following which they will emit light at anotherwavelength, the excitation spectra of the subject proteins typicallyranges from about 300 to 700, usually from about 350 to 650 and moreusually from about 400 to 600 nm while the emission spectra of thesubject proteins typically ranges from about 400 to 800, usually fromabout 425 to 775 and more usually from about 450 to 750 nm. The subjectproteins generally have a maximum extinction coefficient that rangesfrom about 10,000 to 50,000 and usually from about 15,000 to 45,000. Thesubject proteins typically range in length from about 150 to 300 andusually from about 200 to 300 amino acid residues, and generally have amolecular weight ranging from about 15 to 35 kDa, usually from about17.5 to 32.5 kDa.

In certain embodiments, the subject proteins are bright, where by brightis meant that the chromoproteins and their fluorescent mutants can bedetected by common methods (e.g., visual screening, spectrophotometry,spectrofluorometry, fluorescent microscopy, by FACS machines, etc.)Fluorescence brightness of particular fluorescent proteins is determinedby its quantum yield multiplied by maximal extinction coefficient.

Brightness of a chromoproteins may be expressed by its maximalextinction coefficient.

In certain embodiments, the subject proteins fold rapidly followingexpression in the host cell. By rapidly folding is meant that theproteins achieve their tertiary structure that gives rise to theirchromo- or fluorescent quality in a short period of time. In theseembodiments, the proteins fold in a period of time that generally doesnot exceed about 3 days, usually does not exceed about 2 days and moreusually does not exceed about 1 day.

Specific proteins of interest are chromo/fluoroproteins (and mutantsthereof) from the following specific Anthozoan species: Anemonia majano,Clavularia sp., Zoanthus sp., Zoanthus sp., Discosoma striata, Discosomasp. “red”, Anemonia sulcata, Discosoma sp “green”, Discosoma sp.“magenta.” Each of these particular types of polypeptide compositions ofinterest is now discussed in greater detail individually.

Anemonia majano (NFP-1; AmCyan)

In many embodiments, the proteins have an absorbance maximum rangingfrom about 250 to 650, usually from about 400 to 500 and more usuallyfrom about 440 to 480 nm while the emission maximum typically rangesfrom about 270 to 670, usually from about 420 to 520 and more usuallyfrom about 460 to 500 nm. The subject proteins typically range in lengthfrom about 200 to 250, usually from about 210 to 240 amino acidresidues, and generally have a molecular weight ranging from about 20 to30, usually from about 22.50 to 27.50 kDa. Of particular interest inmany embodiments is amFP486 (NFP-1), which has an amino acid sequence asshown in SEQ ID NO:02. Also of interest are mutants of this sequence,where specific mutants of interest include, but are not limited to:Mut15, Mut32, and FP1-NA (a non-aggregating mutant), where thesespecific mutants are further described in the experimental section,infra.

Clavularia sp. (NFP-2)

In certain embodiments, the proteins have an absorbance maximum thattypically ranges from about 250 to 650, usually from about 400 to 500and more usually from about 440 to 480 nm and an emission maximum thattypically ranges from about 270 to 670, usually from about 420 to 520and more usually from about 460 to 500 nm, where the subject proteinstypically range in length from about 225 to 300, usually from about 250to 275 amino acid residues, and generally have a molecular weightranging from about 25 to 35, usually from about 27.50 to 32.50 kDa. Ofparticular interest is the cFP484 protein having the sequence shown inSEQ ID NO:04. Specific mutants of interest include, but are not limitedto: Δ19 cFP484 and Δ38 cFP484, where these specific mutants are furtherdescribed in the experimental section, infra.

Zoanthus sp I. (NFP-3; ZsGreen)

In many embodiments, the proteins have an absorbance maximum thattypically ranges from about 300 to 700, usually from about 450 to 550and more usually from about 480 to 510 nm and an emission maximum thattypically ranges from about 320 to 720, usually from about 470 to 570and more usually from about 500 to 530 nm. The subject proteinstypically range in length from about 200 to 250, usually from about 220to 240 amino acid residues, and generally have a molecular weightranging from about 20 to 30, usually from about 22.50 to 27.50 kDa. Ofparticular interest is the protein zFP506 (NFP-3) which has an aminoacid sequence as shown in SEQ ID NO:06. Specific mutants of interestinclude, but are not limited to: N66M; FP-3NA (a non-aggregatingmutant); yellow; yellow bright, etc., where these specific mutants arefurther described in the experimental section, infra.

Zoanthus sp. II (NFP-4; ZsYellow)

In many embodiments, the proteins have an excitation maximum thattypically ranges from about 300 to 650, usually from about 475 to 575and more usually from about 500 to 550 nm and an emission maximum thattypically ranges from about 310 to 660, usually from about 485 to 585and more usually from about 510 to 560 nm. The subject proteinstypically range in length from about 200 to 250, usually from about 220to 240 amino acid residues, and generally have a molecular weightranging from about 20 to 30, usually from about 22.50 to 27.50 kDa.Specific mutants of interest include, but are not limited to: M129V;NFP4-NA (a non-aggregating mutant); green; etc., where these specificmutants are further described in the experimental section, infra.

Discosoma striata (NFP-5)

In many embodiments, the proteins have an excitation maximum thattypically ranges from about 240 to 640, usually from about 500 to 600and more usually from about 530 to 560 nm and an emission maximum thattypically ranges from about 280 to 680, usually from about 540 to 640and more usually from about 570 to 600 nm. The subject proteinstypically range in length from about 200 to 250, usually from about 220to 240 amino acid residues, and generally have a molecular weightranging from about 20 to 30, usually from about 22.50 to 27.50 kDa. Ofparticular interest in many embodiments is the protein dsFP483 (NFP-5)which has an amino acid sequence as shown in SEQ ID NO:10, as well asmutants thereof.

Discosoma sp. “red” (NFP-6; DsRed; DsRed2)

In many embodiments, the proteins have an absorbance maximum thattypically ranges from about 250 to 750, usually from about 500 to 600and more usually from about 540 to 580 nm and have an emission maximumthat typically ranges from about 275 to 775, usually from about 525 to625 and more usually from about 565 to 605 nm. The subject proteinstypically range in length from about 200 to 250, usually from about 220to 240 amino acid residues, and generally have a molecular weightranging from about 20 to 30, usually from about 22.50 to 27.50 kDa. Ofparticular interest is the drFP583 (NFP-6) protein that has an aminoacid sequence as shown in SEQ ID NO:12. Specific mutants of interestinclude, but are not limited to: E5, E5-NA (a non-aggregating mutant),E8, E5up, E5down, E57, FP6-NA (a non-aggregating mutant), AG4, AG45,E83, 6/9 Q, 6/9 Q-NA, 6/92G, etc., where these specific mutants arefurther described in the experimental section, infra.

Anemonia sulcata (NFP-7, AsRed)

In many embodiments, the proteins have an absorbance maximum thattypically ranges from about 370 to 770, usually from about 520 to 620and more usually from about 560 to 580 nm and an emission maximum thattypically ranges from about 395 to 795, usually from about 545 to 645and more usually from about 585 to 605 nm. The subject proteinstypically range in length from about 200 to 250, usually from about 220to 240 amino acid residues, and generally have a molecular weightranging from about 20 to 30, usually from about 22.50 to 27.50 kDa. Ofparticular interest is the asFP595(asFP600) (NFP-7) protein that has anamino acid sequence as shown in SEQ ID NO:14. Specific mutants ofinterest include, but are not limited to: Mut1; Mut35-5/Mut1; FP7-NA (anon-aggregating mutant), etc., where these specific mutants are furtherdescribed in the experimental section, infra.

Discosoma sp “green” (NFP-8)

In many embodiments, the proteins have an absorbance maximum thattypically ranges from about 300 to 700, usually from about 450 to 650and more usually from about 490 to 510 nm and an emission maximum thattypically ranges from about 310 to 710, usually from about 460 to 660and more usually from about 500 to 520 nm. The subject proteinstypically range in length from about 200 to 250, usually from about 220to 240 amino acid residues, and generally have a molecular weightranging from about 20 to 30, usually from about 22.50 to 27.50 kDa. Ofparticular interest is the dgFP512 protein (NFP-8) protein that has anamino acid sequence as shown in SEQ ID NO:16, as well as mutantsthereof.

Discosoma sp. “magenta” (NFP-9)

In many embodiments, the proteins have an absorbance maximum thattypically ranges from about 375 to 775, usually from about 525 to 625and more usually from about 560 to 590 nm and an emission maximum thattypically ranges from about 395 to 795, usually from about 545 to 645and more usually from about 580 to 610 nm. The subject proteinstypically range in length from about 200 to 250, usually from about 220to 240 amino acid residues, and generally have a molecular weightranging from about 20 to 30, usually from about 22.50 to 27.50 kDa. Ofparticular interest is the dmFP592 (NFP-9) protein that has an aminoacid sequence as shown in SEQ ID NO:18, as well as mutants thereof.

Homologs or proteins (or fragments thereof) that vary in sequence fromthe above provided specific amino acid sequences of the subjectinvention, i.e., SEQ ID NOS: 02; 04; 06; 08; 10; 12; 14;16 or 18, arealso provided. By homolog is meant a protein having at least about 10%,usually at least about 20% and more usually at least about 30%, and inmany embodiments at least about 35%, usually at least about 40% and moreusually at least about 60% amino acid sequence identity to the proteinof the subject invention, as determined using MegAlign, DNAstar (1998)clustal algorithm as described in D. G. Higgins and P. M. Sharp, “Fastand Sensitive multiple Sequence Alignments on a Microcomputer,” (1989)CABIOS, 5: 151–153. (Parameters used are ktuple 1, gap penalty 3,window, 5 and diagonals saved 5). In many embodiments, homologues ofinterest have much higher sequence identify, e.g., 65%, 70%, 75%, 80%,85%, 90% or higher.

Also provided are proteins that are substantially identical to the wildtype protein, where by substantially identical is meant that the proteinhas an amino acid sequence identity to the sequence of wild type proteinof at least about 60%, usually at least about 65% and more usually atleast about 70%, where in some instances the identity may be muchhigher, e.g., 75%, 80%, 85%, 90%, 95% or higher.

In many embodiments, the subject homologues have structural featuresfound in the above provided specific sequences, where such structuralfeatures include the β-can fold.

Proteins which are mutants of the above-described naturally occurringproteins are also provided. Mutants may retain biological properties ofthe wild-type (e.g., naturally occurring) proteins, or may havebiological properties which differ from the wild-type proteins. The term“biological property” of the subject proteins includes, but is notlimited to, spectral properties, such as absorbance maximum, emissionmaximum, maximum extinction coefficient, brightness (e.g., as comparedto the wild-type protein or another reference protein such as greenfluorescent protein from A. victoria), and the like; in vivo and/or invitro stability (e.g., half-life); etc. Mutants include single aminoacid changes, deletions of one or more amino acids, N-terminaltruncations, C-terminal truncations, insertions, etc.

Mutants can be generated using standard techniques of molecular biology,e.g., random mutagenesis, and targeted mutagenesis. Several mutants aredescribed herein. Given the guidance provided in the Examples, and usingstandard techniques, those skilled in the art can readily generate awide variety of additional mutants and test whether a biologicalproperty has been altered. For example, fluorescence intensity can bemeasured using a spectrophotometer at various excitation wavelengths.

Those proteins of the subject invention that are naturally occurringproteins are present in a non-naturally occurring environment, e.g., areseparated from their naturally occurring environment. In certainembodiments, the subject proteins are present in a composition that isenriched for the subject protein as compared to its naturally occurringenvironment. For example, purified protein is provided, where bypurified is meant that the protein is present in a composition that issubstantially free of non-chromo/fluoroprotein proteins of interest,where by substantially free is meant that less than 90%, usually lessthan 60% and more usually less than 50% of the composition is made up ofnon-chromoproteins or mutants thereof of interest. The proteins of thesubject invention may also be present as an isolate, by which is meantthat the protein is substantially free of other proteins and othernaturally occurring biologic molecules, such as oligosaccharides,polynucleotides and fragments thereof, and the like, where the term“substantially free” in this instance means that less than 70%, usuallyless than 60% and more usually less than 50% of the compositioncontaining the isolated protein is some other naturally occurringbiological molecule. In certain embodiments, the proteins are present insubstantially pure form, where by “substantially pure form” is meant atleast 95%, usually at least 97% and more usually at least 99% pure.

In addition to the naturally occurring proteins, polypeptides that varyfrom the naturally occurring proteins, e.g., the mutant proteinsdescribed above, are also provided. Generally such polypeptides includean amino acid sequence encoded by an open reading frame (ORF) of thegene encoding the subject wild type protein, including the full lengthprotein and fragments thereof, particularly biologically activefragments and/or fragments corresponding to functional domains, and thelike; and including fusions of the subject polypeptides to otherproteins or parts thereof. Fragments of interest will typically be atleast about 10 aa in length, usually at least about 50 aa in length, andmay be as long as 300 aa in length or longer, but will usually notexceed about 1000 aa in length, where the fragment will have a stretchof amino acids that is identical to the subject protein of at leastabout 10 aa, and usually at least about 15 aa, and in many embodimentsat least about 50 aa in length. In some embodiments, the subjectpolypeptides are about 25 aa, about 50 aa, about 75 aa, about 100 aa,about 125 aa, about 150 aa, about 200 aa, about 210 aa, about 220 aa,about 230 aa, or about 240 aa in length, up to the entire protein. Insome embodiments, a protein fragment retains all or substantially all ofa biological property of the wild-type protein.

The subject proteins and polypeptides may be obtained from naturallyoccurring sources or synthetically produced. For example, wild typeproteins may be derived from biological sources which express theproteins, e.g., non-bioluminescent Cnidarian, e.g., Anthozoan, species,such as the specific ones listed above. The subject proteins may also bederived from synthetic means, e.g., by expressing a recombinant gene ornucleic acid coding sequence encoding the protein of interest in asuitable host, as described above. Any convenient protein purificationprocedures may be employed, where suitable protein purificationmethodologies are described in Guide to Protein Purification, (Deuthsered.) (Academic Press, 1990). For example, a lysate may prepared from theoriginal source and purified using HPLC, exclusion chromatography, gelelectrophoresis, affinity chromatography, and the like.

Antibody Compositions

Also provided are antibodies that specifically bind to the subjectfluorescent proteins. Suitable antibodies are obtained by immunizing ahost animal with peptides comprising all or a portion of the subjectprotein. Suitable host animals include mouse, rat sheep, goat, hamster,rabbit, etc. The origin of the protein immunogen will generally be aCnidarian species, specifcally a non-bioluminescent Cnidarian species,such as an Anthozoan species or a non-Petalucean Anthozoan species. Thehost animal will generally be a different species than the immunogen,e.g., mice, etc.

The immunogen may comprise the complete protein, or fragments andderivatives thereof. Preferred immunogens comprise all or a part of theprotein, where these residues contain the post-translation modificationsfound on the native target protein. Immunogens are produced in a varietyof ways known in the art, e.g., expression of cloned genes usingconventional recombinant methods, isolation from Anthozoan species oforigin, etc.

For preparation of polyclonal antibodies, the first step is immunizationof the host animal with the target protein, where the target proteinwill preferably be in substantially pure form, comprising less thanabout 1% contaminant. The immunogen may comprise the complete targetprotein, fragments or derivatives thereof. To increase the immuneresponse of the host animal, the target protein may be combined with anadjuvant, where suitable adjuvants include alum, dextran, sulfate, largepolymeric anions, oil & water emulsions, e.g. Freund's adjuvant,Freund's complete adjuvant, and the like. The target protein may also beconjugated to synthetic carrier proteins or synthetic antigens. Avariety of hosts may be immunized to produce the polyclonal antibodies.Such hosts include rabbits, guinea pigs, rodents, e.g. mice, rats,sheep, goats, and the like. The target protein is administered to thehost, usually intradermally, with an initial dosage followed by one ormore, usually at least two, additional booster dosages. Followingimmunization, the blood from the host will be collected, followed byseparation of the serum from the blood cells. The Ig present in theresultant antiserum may be further fractionated using known methods,such as ammonium salt fractionation, DEAE chromatography, and the like.

Monoclonal antibodies are produced by conventional techniques.Generally, the spleen and/or lymph nodes of an immunized host animalprovide a source of plasma cells. The plasma cells are immortalized byfusion with myeloma cells to produce hybridoma cells. Culturesupernatant from individual hybridomas is screened using standardtechniques to identify those producing antibodies with the desiredspecificity. Suitable animals for production of monoclonal antibodies tothe human protein include mouse, rat, hamster, etc. To raise antibodiesagainst the mouse protein, the animal will generally be a hamster,guinea pig, rabbit, etc. The antibody may be purified from the hybridomacell supernatants or ascites fluid by conventional techniques, e.g.affinity chromatography using protein bound to an insoluble support,protein A sepharose, etc.

The antibody may be produced as a single chain, instead of the normalmultimeric structure. Single chain antibodies are described in Jost etal. (1994) J.B.C. 269:26267–73, and others. DNA sequences encoding thevariable region of the heavy chain and the variable region of the lightchain are ligated to a spacer encoding at least about 4 amino acids ofsmall neutral amino acids, including glycine and/or serine. The proteinencoded by this fusion allows assembly of a functional variable regionthat retains the specificity and affinity of the original antibody.

Also of interest in certain embodiments are humanized antibodies.Methods of humanizing antibodies are known in the art. The humanizedantibody may be the product of an animal having transgenic humanimmunoglobulin constant region genes (see for example InternationalPatent Applications WO 90/10077 and WO 90/04036). Alternatively, theantibody of interest may be engineered by recombinant DNA techniques tosubstitute the CH1, CH2, CH3, hinge domains, and/or the framework domainwith the corresponding human sequence (see WO 92/02190).

The use of Ig cDNA for construction of chimeric immunoglobulin genes isknown in the art (Liu et al. (1987) P.N.A.S. 84:3439 and (1987) J.Immunol. 139:3521). mRNA is isolated from a hybridoma or other cellproducing the antibody and used to produce cDNA. The cDNA of interestmay be amplified by the polymerase chain reaction using specific primers(U.S. Pat. Nos. 4,683,195 and 4,683,202). Alternatively, a library ismade and screened to isolate the sequence of interest. The DNA sequenceencoding the variable region of the antibody is then fused to humanconstant region sequences. The sequences of human constant regions genesmay be found in Kabat et al. (1991) Sequences of Proteins ofImmunological Interest, N.I.H. publication no. 91-3242. Human C regiongenes are readily available from known clones. The choice of isotypewill be guided by the desired effector functions, such as complementfixation, or activity in antibody-dependent cellular cytotoxicity.Preferred isotypes are IgG1, IgG3 and IgG4. Either of the human lightchain constant regions, kappa or lambda, may be used. The chimeric,humanized antibody is then expressed by conventional methods.

Antibody fragments, such as Fv, F(ab′)₂ and Fab may be prepared bycleavage of the intact protein, e.g. by protease or chemical cleavage.Alternatively, a truncated gene is designed. For example, a chimericgene encoding a portion of the F(ab′)₂ fragment would include DNAsequences encoding the CH1 domain and hinge region of the H chain,followed by a translational stop codon to yield the truncated molecule.

Consensus sequences of H and L J regions may be used to designoligonucleotides for use as primers to introduce useful restrictionsites into the J region for subsequent linkage of V region segments tohuman C region segments. C region cDNA can be modified by site directedmutagenesis to place a restriction site at the analogous position in thehuman sequence.

Expression vectors include plasmids, retroviruses, YACs, EBV derivedepisomes, and the like. A convenient vector is one that encodes afunctionally complete human CH or CL immunoglobulin sequence, withappropriate restriction sites engineered so that any VH or VL sequencecan be easily inserted and expressed. In such vectors, splicing usuallyoccurs between the splice donor site in the inserted J region and thesplice acceptor site preceding the human C region, and also at thesplice regions that occur within the human CH exons. Polyadenylation andtranscription termination occur at native chromosomal sites downstreamof the coding regions. The resulting chimeric antibody may be joined toany strong promoter, including retroviral LTRs, e.g. SV-40 earlypromoter, (Okayama et al. (1983) Mol. Cell. Bio. 3:280), Rous sarcomavirus LTR (Gorman et al. (1982) P.N.A.S. 79:6777), and moloney murineleukemia virus LTR (Grosschedl et al. (1985) Cell 41:885); native Igpromoters, etc.

Transgenics

The subject nucleic acids can be used to generate transgenic, non-humanplants or animals or site specific gene modifications in cell lines.Transgenic cells of the subject invention include on or more nucleicacids according to the subject invention present as a transgene, whereincluded within this definition are the parent cells transformed toinclude the transgene and the progeny thereof. In many embodiments, thetransgenic cells are cells that do not normally harbor or contain anucleic acid according to the subject invention. In those embodimentswhere the transgenic cells do naturally contain the subject nucleicacids, the nucleic acid will be present in the cell in a position otherthan its natural location, i.e. integrated into the genomic material ofthe cell at a non-natural location. Transgenic animals may be madethrough homologous recombination, where the endogenous locus is altered.Alternatively, a nucleic acid construct is randomly integrated into thegenome. Vectors for stable integration include plasmids, retrovirusesand other animal viruses, YACs, and the like.

Transgenic organisms of the subject invention include cells andmulticellular organisms, e.g., plants and animals, that are endogenousknockouts in which expression of the endogenous gene is at least reducedif not eliminated. Transgenic organisms of interest also include cellsand multicellular organisms, e.g., plants and animals, in which theprotein or variants thereof is expressed in cells or tissues where it isnot normally expressed and/or at levels not normally present in suchcells or tissues.

DNA constructs for homologous recombination will comprise at least aportion of the gene of the subject invention, wherein the gene has thedesired genetic modification(s), and includes regions of homology to thetarget locus. DNA constructs for random integration need not includeregions of homology to mediate recombination. Conveniently, markers forpositive and negative selection are included. Methods for generatingcells having targeted gene modifications through homologousrecombination are known in the art. For various techniques fortransfecting mammalian cells, see Keown et al. (1990), Meth. Enzymol.185:527–537.

For embryonic stem (ES) cells, an ES cell line may be employed, orembryonic cells may be obtained freshly from a host, e.g. mouse, rat,guinea pig, etc. Such cells are grown on an appropriatefibroblast-feeder layer or grown in the presence of leukemia inhibitingfactor (LIF). When ES or embryonic cells have been transformed, they maybe used to produce transgenic animals. After transformation, the cellsare plated onto a feeder layer in an appropriate medium. Cellscontaining the construct may be detected by employing a selectivemedium. After sufficient time for colonies to grow, they are picked andanalyzed for the occurrence of homologous recombination or integrationof the construct. Those colonies that are positive may then be used forembryo manipulation and blastocyst injection. Blastocysts are obtainedfrom 4 to 6 week old superovulated females. The ES cells aretrypsinized, and the modified cells are injected into the blastocoel ofthe blastocyst. After injection, the blastocysts are returned to eachuterine horn of pseudopregnant females. Females are then allowed to goto term and the resulting offspring screened for the construct. Byproviding for a different phenotype of the blastocyst and thegenetically modified cells, chimeric progeny can be readily detected.

The chimeric animals are screened for the presence of the modified geneand males and females having the modification are mated to producehomozygous progeny. If the gene alterations cause lethality at somepoint in development, tissues or organs can be maintained as allogeneicor congenic grafts or transplants, or in in vitro culture. Thetransgenic animals may be any non-human mammal, such as laboratoryanimals, domestic animals, etc. The transgenic animals may be used infunctional studies, drug screening, etc. Representative examples of theuse of transgenic animals include those described infra.

Transgenic plants may be produced in a similar manner. Methods ofpreparing transgenic plant cells and plants are described in U.S. Pat.Nos. 5,767,367; 5,750,870; 5,739,409; 5,689,049; 5,689,045; 5,674,731;5,656,466; 5,633,155; 5,629,470; 5,595,896; 5,576,198; 5,538,879;5,484,956; the disclosures of which are herein incorporated byreference. Methods of producing transgenic plants are also reviewed inPlant Biochemistry and Molecular Biology (eds Lea & Leegood, John Wiley& Sons)(1993) pp 275–295. In brief, a suitable plant cell or tissue isharvested, depending on the nature of the plant species. As such, incertain instances, protoplasts will be isolated, where such protoplastsmay be isolated from a variety of different plant tissues, e.g. leaf,hypoctyl, root, etc. For protoplast isolation, the harvested cells areincubated in the presence of cellulases in order to remove the cellwall, where the exact incubation conditions vary depending on the typeof plant and/or tissue from which the cell is derived. The resultantprotoplasts are then separated from the resultant cellular debris bysieving and centrifugation. Instead of using protoplasts, embryogenicexplants comprising somatic cells may be used for preparation of thetransgenic host. Following cell or tissue harvesting, exogenous DNA ofinterest is introduced into the plant cells, where a variety ofdifferent techniques are available for such introduction. With isolatedprotoplasts, the opportunity arise for introduction via DNA-mediatedgene transfer protocols, including: incubation of the protoplasts withnaked DNA, e.g. plasmids, comprising the exogenous coding sequence ofinterest in the presence of polyvalent cations, e.g. PEG or PLO; andelectroporation of the protoplasts in the presence of naked DNAcomprising the exogenous sequence of interest. Protoplasts that havesuccessfully taken up the exogenous DNA are then selected, grown into acallus, and ultimately into a transgenic plant through contact with theappropriate amounts and ratios of stimulatory factors, e.g. auxins andcytokinins. With embryogenic explants, a convenient method ofintroducing the exogenous DNA in the target somatic cells is through theuse of particle acceleration or “gene-gun” protocols. The resultantexplants are then allowed to grow into chimera plants, cross-bred andtransgenic progeny are obtained. Instead of the naked DNA approachesdescribed above, another convenient method of producing transgenicplants is Agrobacterium mediated transformation. With Agrobacteriummediated transformation, co-integrative or binary vectors comprising theexogenous DNA are prepared and then introduced into an appropriateAgrobacterium strain, e.g. A. tumefaciens. The resultant bacteria arethen incubated with prepared protoplasts or tissue explants, e.g. leafdisks, and a callus is produced. The callus is then grown underselective conditions, selected and subjected to growth media to induceroot and shoot growth to ultimately produce a transgenic plant.

Utility

The subject chromoproteins and fluorescent mutants thereof find use in avariety of different applications, where the applications necessarilydiffer depending on whether the protein is a chromoprotein or afluorescent protein. Representative uses for each of these types ofproteins will be described below, where the follow described uses aremerely representative and are in no way meant to limit the use of thesubject proteins to those described below.

Chromoproteins

The subject chromoproteins of the present invention find use in avariety of different applications. One application of interest is theuse of the subject proteins as coloring agents which are capable ofimparting color or pigment to a particular composition of matter. Ofparticular interest in certain embodiments are non-toxic chromoproteins.The subject chromoproteins may be incorporated into a variety ofdifferent compositions of matter, where representative compositions ofmatter include: food compositions, pharmaceuticals, cosmetics, livingorganisms, e.g., animals and plants, and the like. Where used as acoloring agent or pigment, a sufficient amount of the chromoprotein isincorporated into the composition of matter to impart the desired coloror pigment thereto. The chromoprotein may be incorporated into thecomposition of matter using any convenient protocol, where theparticular protocol employed will necessarily depend, at least in part,on the nature of the composition of matter to be colored. Protocols thatmay be employed include, but are not limited to: blending, diffusion,friction, spraying, injection, tattooing, and the like.

The chromoproteins may also find use as labels in analyte detectionassays, e.g., assays for biological analytes of interest. For example,the chromoproteins may be incorporated into adducts with analytespecific antibodies or binding fragments thereof and subsequentlyemployed in immunoassays for analytes of interest in a complex sample,as described in U.S. Pat. No. 4,302,536; the disclosure of which isherein incorporated by reference. Instead of antibodies or bindingfragments thereof, the subject chromoproteins or chromogenic fragmentsthereof may be conjugated to ligands that specifically bind to ananalyte of interest, or other moieties, growth factors, hormones, andthe like; as is readily apparent to those of skill in the art.

In yet other embodiments, the subject chromoproteins may be used asselectable markers in recombinant DNA applications, e.g., the productionof transgenic cells and organisms, as described above. As such, one canengineer a particular transgenic production protocol to employexpression of the subject chromoproteins as a selectable marker, eitherfor a successful or unsuccessful protocol. Thus, appearance of the colorof the subject chromoprotein in the phenotype of the transgenic organismproduced by a particular process can be used to indicate that theparticular organism successfully harbors the transgene of interest,often integrated in a manner that provides for expression of thetransgene in the organism. When used a selectable marker, a nucleic acidencoding for the subject chromoprotein can be employed in the transgenicgeneration process, where this process is described in greater detailsupra. Particular transgenic organisms of interest where the subjectproteins may be employed as selectable markers include transgenicplants, animals, bacteria, fungi, and the like.

In yet other embodiments, the chromoproteins (and fluorescent proteins)of the subject invention find use in sunscreens, as selective filters,etc., in a manner similar to the uses of the proteins described in WO00/46233.

Fluorescent Proteins

The subject fluorescent proteins of the present invention (as well asother components of the subject invention described above) find use in avariety of different applications, where such applications include, butare not limited to, the following. The first application of interest isthe use of the subject proteins in fluorescence resonance energytransfer (FRET) applications. In these applications, the subjectproteins serve as donor and/or acceptors in combination with a secondfluorescent protein or dye, e.g., a fluorescent protein as described inMatz et al., Nature Biotechnology (October 1999) 17:969–973, a greenfluorescent protein from Aequoria victoria or fluorescent mutantthereof, e.g., as described in U.S. Pat. Nos. 6,066,476; 6,020,192;5,985,577; 5,976,796; 5,968,750; 5,968,738; 5,958,713; 5,919,445;5,874,304, the disclosures of which are herein incorporated byreference, other fluorescent dyes, e.g., coumarin and its derivatives,e.g. 7-amino-4-methylcoumarin, aminocoumarin, bodipy dyes, such asBodipy FL, cascade blue, fluorescein and its derivatives, e.g.fluorescein isothiocyanate, Oregon green, rhodamine dyes, e.g. texasred, tetramethylrhodamine, eosins and erythrosins, cyanine dyes, e.g.Cy3 and Cy5, macrocyclic chelates of lanthamide ions, e.g. quantum dye,etc., chemilumescent dyes, e.g., luciferases, including those describedin U.S. Pat. Nos. 5,843,746; 5,700,673; 5,674,713; 5,618,722; 5,418,155;5,330,906; 5,229,285; 5,221,623; 5,182,202; the disclosures of which areherein incorporated by reference. Specific examples of where FRET assaysemploying the subject fluorescent proteins may be used include, but arenot limited to: the detection of protein-protein interactions, e.g.,mammalian two-hybrid system, transcription factor dimerization, membraneprotein multimerization, multiprotein complex formation, etc., as abiosensor for a number of different events, where a peptide or proteincovalently links a FRET fluorescent combination including the subjectfluorescent proteins and the linking peptide or protein is, e.g., aprotease specific substrate, e.g., for caspase mediated cleavage, alinker that undergoes conformational change upon receiving a signalwhich increases or decreases FRET, e.g., PKA regulatory domain(cAMP-sensor), phosphorylation, e.g., where there is a phosphorylationsite in the linker or the linker has binding specificity tophosphorylated/dephosphorylated domain of another protein, or the linkerhas Ca²⁺binding domain. Representative fluorescence resonance energytransfer or FRET applications in which the subject proteins find useinclude, but are not limited to, those described in: U.S. Pat. Nos.6,008,373; 5,998,146; 5,981,200; 5,945,526; 5,945,283; 5,911,952;5,869,255; 5,866,336; 5,863,727; 5,728,528; 5,707,804; 5,688,648;5,439,797; the disclosures of which are herein incorporated byreference.

The subject fluorescent proteins also find use as biosensors inprokaryotic and eukaryotic cells, e.g. as Ca²+ion indicator; as pHindicator, as phorphorylation indicator, as an indicator of other ions,e.g., magnesium, sodium, potassium, chloride and halides. For example,for detection of Ca²⁺ ion, proteins containing an EF-hand motif areknown to translocate from the cytosol to membranes upon Ca²⁺ binding.These proteins contain a myristoyl group that is buried within themolecule by hydrophobic interactions with other regions of the protein.Binding of Ca²⁺ induces a conformational change exposing the myristoylgroup which then is available for the insertion into the lipid bilayer(called a “Ca²⁺-myristoyl switch”). Fusion of such a EF-hand containingprotein to Fluorescent Proteins (FP) could make it an indicator ofintracellular Ca²⁺ by monitoring the translocation from the cytosol tothe plasma membrane by confocal microscopy. EF-hand proteins suitablefor use in this system include, but are not limited to: recoverin (1–3),calcineurin B, troponin C, visinin, neurocalcin, calmodulin,parvalbumin, and the like. For pH, a system based on hisactophilins maybe employed. Hisactophilins are myristoylated histidine-rich proteinsknown to exist in Dictyostelium. Their binding to actin and acidiclipids is sharply pH-dependent within the range of cytoplasmic pHvariations. In living cells membrane binding seems to override theinteraction of hisactophilins with actin filaments. At pH 6.5 theylocate to the plasma membrane and nucleus. In contrast, at pH 7.5 theyevenly distribute throughout the cytoplasmic space. This change ofdistribution is reversible and is attributed to histidine clustersexposed in loops on the surface of the molecule. The reversion ofintracellular distribution in the range of cytoplasmic pH variations isin accord with a pK of 6.5 of histidine residues. The cellulardistribution is independent of myristoylation of the protein. By fusingFPs (Fluoresent Proteins) to hisactophilin the intracellulardistribution of the fusion protein can be followed by laser scanning,confocal microscopy or standard fluorescence microscopy. Quantitativefluorescence analysis can be done by performing line scans through cells(laser scanning confocal microscopy) or other electronic data analysis(e.g., using metamorph software (Universal Imaging Corp) and averagingof data collected in a population of cells. Substantial pH-dependentredistribution of hisactophilin-FP from the cytosol to the plasmamembrane occurs within 1–2 min and reaches a steady state level after5–10 min. The reverse reaction takes place on a similar time scale. Assuch, hisactophilin-fluorescent protein fusion protein that acts in ananalogous fashion can be used to monitor cytosolic pH changes in realtime in live mammalian cells. Such methods have use in high throuhgputapplications, e.g., in the measurement of pH changes as consequence ofgrowth factor receptor activation (e.g. epithelial or platelet-derivedgrowth factor) chemotactic stimulation/cell locomotion, in the detectionof intracellular pH changes as second messenger, in the monitoring ofintracellular pH in pH manipulating experiments, and the like. Fordetection of PKC activity, the reporter system exploits the fact that amolecule called MARCKS (myristoylated alanine-rich C kinase substrate)is a PKC substrate. It is anchored to the plasma membrane viamyristoylation and a stretch of positively charged amino acids(ED-domain) that bind to the negatively charged plasma membrane viaelectrostatic interactions. Upon PKC activation the ED-domain becomesphosphorylated by PKC, thereby becoming negatively charged, and as aconsequence of electrostatic repulsion MARCKS translocates from theplasma membrane to the cytoplasm (called the “myristoyl-electrostaticswitch”). Fusion of the N-terminus of MARCKS ranging from themyristoylation motif to the ED-domain of MARCKS to fluorescent proteinsof the present invention makes the above a detector system for PKCactivity. When phosphorylated by PKC, the fusion protein translocatesfrom the plasma membrane to the cytosol. This translocation is followedby standard fluorescence microscopy or confocal microscopy e.g. usingthe Cellomics technology or other High Content Screening systems (e.g.Universal Imaging Corp./Becton Dickinson). The above reporter system hasapplication in High Content Screening, e.g., screening for PKCinhibitors, and as an indicator for PKC activity in many screeningscenarios for potential reagents interfering with this signaltransduction pathway. Methods of using fluorescent proteins asbiosensors also include those described in U.S. Pat. Nos. 972,638;5,824,485 and 5,650,135 (as well as the references cited therein) thedisclosures of which are herein incorporated by reference.

The subject fluorescent proteins also find use in applications involvingthe automated screening of arrays of cells expressing fluorescentreporting groups by using microscopic imaging and electronic analysis.Screening can be used for drug discovery and in the field of functionalgenomics: e.g., where the subject proteins are used as markers of wholecells to detect changes in multicellular reorganization and migration,e.g., formation of multicellular tubules (blood vessel formation) byendothelial cells, migration of cells through Fluoroblok Insert System(Becton Dickinson Co.), wound healing, neurite outgrowth, etc.; wherethe proteins are used as markers fused to peptides (e.g., targetingsequences) and proteins that allow the detection of change ofintracellular location as indicator for cellular activity, for example:signal transduction, such as kinase and transcription factortranslocation upon stimuli, such as protein kinase C, protein kinase A,transcription factor NFkB, and NFAT; cell cycle proteins, such as cyclinA, cyclin B1 and cyclinE; protease cleavage with subsequent movement ofcleaved substrate, phospholipids, with markers for intracellularstructures such as endoplasmic reticulum, Golgi apparatus, mitochondria,peroxisomes, nucleus, nucleoli, plasma membrane, histones, endosomes,lysosomes, microtubules, actin) as tools for High Content Screening:co-localization of other fluorescent fusion proteins with theselocalization markers as indicators of movements of intracellularfluorescent fusion proteins/peptides or as marker alone; and the like.Examples of applications involving the automated screening of arrays ofcells in which the subject fluorescent proteins find use include: U.S.Pat. No. 5,989,835; as well as WO/0017624; WO 00/26408; WO 00/17643; andWO 00/03246; the disclosures of which are herein incorporated byreference.

The subject fluorescent proteins also find use in high through-putscreening assays. The subject fluorescent proteins are stable proteinswith half-lives of more than 24 h. Also provided are destabilizedversions of the subject fluorescent proteins with shorter half-livesthat can be used as transcription reporters for drug discovery. Forexample, a protein according to the subject invention can be fused witha putative proteolytic signal sequence derived from a protein withshorter half-life, e.g., PEST sequence from the mouse ornithinedecarboxylase gene, mouse cyclin B1 destruction box and ubiquitin, etc.For a description of destabilized proteins and vectors that can beemployed to produce the same, see e.g., U.S. Pat. No. 6,130,313; thedisclosure of which is herein incorporated by reference. Promoters insignal transduction pathways can be detected using destabilized versionsof the subject fluorescent proteins for drug screening, e.g., AP1, NFAT,NFkB, Smad, STAT, p53, E2F, Rb, myc, CRE, ER, GR and TRE, and the like.

The subject proteins can be used as second messenger detectors, e.g., byfusing the subject proteins to specific domains: e.g., PKCgamma Cabinding domain, PKCgamma DAG binding domain, SH2 domain and SH3 domain,etc.

Secreted forms of the subject proteins can be prepared, e.g. by fusingsecreted leading sequences to the subject proteins to construct secretedforms of the subject proteins, which in turn can be used in a variety ofdifferent applications.

The subject proteins also find use in fluorescence activated cellsorting applications. In such applications, the subject fluorescentprotein is used as a label to mark a population of cells and theresulting labeled population of cells is then sorted with a fluorescentactivated cell sorting device, as is known in the art. FACS methods aredescribed in U.S. Pat. Nos. 5,968,738 and 5,804,387; the disclosures ofwhich are herein incorporated by reference.

The subject proteins also find use as in vivo marker in animals (e.g.,transgenic animals). For example, expression of the subject protein canbe driven by tissue specific promoters, where such methods find use inresearch for gene therapy, e.g., testing efficiency of transgenicexpression, among other applications. A representative application offluorescent proteins in transgenic animals that illustrates this classof applications of the subject proteins is found in WO 00/02997, thedisclosure of which is herein incorporated by reference.

Additional applications of the subject proteins include: as markersfollowing injection into cells or animals and in calibration forquantitative measurements (fluorescence and protein); as markers orreporters in oxygen biosensor devices for monitoring cell viability; asmarkers or labels for animals, pets, toys, food, etc.; and the like.

The subject fluorescent proteins also find use in protease cleavageassays. For example, cleavage inactivated fluorescence assays can bedeveloped using the subject proteins, where the subject proteins areengineered to include a protease specific cleavage sequence withoutdestroying the fluorescent character of the protein. Upon cleavage ofthe fluorescent protein by an activated protease fluorescence wouldsharply decrease due to the destruction of a functional chromophor.Alternatively, cleavage activated fluorescence can be developed usingthe subject proteins, where the subject proteins are engineered tocontain an additional spacer sequence in close proximity/or inside thechromophor. This variant would be significantly decreased in itsfluorescent activity, because parts of the functional chromophor wouldbe divided by the spacer. The spacer would be framed by two identicalprotease specific cleavage sites. Upon cleavage via the activatedprotease the spacer would be cut out and the two residual “subunits” ofthe fluorescent protein would be able to reassemble to generate afunctional fluorescent protein. Both of the above types of applicationcould be developed in assays for a variety of different types ofproteases, e.g., caspases, etc.

The subject proteins can also be used is assays to determine thephospholipid composition in biological membranes. For example, fusionproteins of the subject proteins (or any other kind of covalent ornon-covalent modification of the subject proteins) that allows bindingto specific phospholipids to localize/visualize patterns of phospholipiddistribution in biological membranes also allowing colocalization ofmembrane proteins in specific phospholipid rafts can be accomplishedwith the subject proteins. For example, the PH domain of GRP1 has a highaffinity to phosphatidyl-inositol tri-phosphate (PIP3) but not to PIP2.As such, a fusion protein between the PH domain of GRP1 and the subjectproteins can be constructed to specifically label PIP3 rich areas inbiological membranes.

Yet another application of the subject proteins is as a fluorescenttimer, in which the switch of one fluorescent color to another (e.g.green to red) concomitant with the ageing of the fluorescent protein isused to determine the activation/deactivation of gene expression, e.g.,developmental gene expression, cell cycle dependent gene expression,circadian rhythm specific gene expression, and the like

The antibodies of the subject invention, described above, also find usein a number of applications, including the differentiation of thesubject proteins from other fluorescent proteins.

Kits

Also provided by the subject invention are kits for use in practicingone or more of the above described applications, where the subject kitstypically include elements for making the subject proteins, e.g., aconstruct comprising a vector that includes a coding region for thesubject protein. The subject kit components are typically present in asuitable storage medium, e.g., buffered solution, typically in asuitable container. Also present in the subject kits may be antibodiesto the provided protein. In certain embodiments, the kit comprises aplurality of different vectors each encoding the subject protein, wherethe vectors are designed for expression in different environments and/orunder different conditions, e.g., constitutive expression where thevector includes a strong promoter for expression in mammalian cells, apromoterless vector with a multiple cloning site for custom insertion ofa promoter and tailored expression, etc.

In addition to the above components, the subject kits will furtherinclude instructions for practicing the subject methods. Theseinstructions may be present in the subject kits in a variety of forms,one or more of which may be present in the kit. One form in which theseinstructions may be present is as printed information on a suitablemedium or substrate, e.g., a piece or pieces of paper on which theinformation is printed, in the packaging of the kit, in a packageinsert, etc. Yet another means would be a computer readable medium,e.g., diskette, CD, etc., on which the information has been recorded.Yet another means that may be present is a website address which may beused via the internet to access the information at a removed site. Anyconvenient means may be present in the kits.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL

I. Wild-Type Anthozoan Proteins

The following table summarizes the properties of nine specific wild typeAnthozoan proteins of the subject invention:

TABLE I Absorb. Emission Maximum Relative Relative Max. Max. ExtinctionQuantum Brightness NFP Species Identifier Nm Nm Coeff. Yield* ** 1Anemonia amFP486 458 486 40,000 0.3 0.43 majano 2 Clavularia sp. cFP484456 484 35,300 0.6 0.77 3 Zoanthus sp. zFP506 496 506 35,600 0.79 1.02 4Zoanthus sp. zFP538 528 538 20,200 0.52 0.38 5 Discosoma dsFP483 443 48323,900 0.57 0.50 striata 6 Discosoma drFP583 558 583 22,500 0.29 0.24sp. “red” 7 Anemonia asFP600 572 596 56,200 <0.001 — sulcata 8 Discosomasp dgFP512 502 512 20,360 0.3 0.21 “green” 9 Discosoma sp. dmFP592 573593 21,800 0.11 0.09 “magenta” *relative quantum yield was determined ascompared to the quantum yield of A. victoria GFP. **relative brightnessis extinction coefficient multiplied by quantum yield divided by thesame value for A. victoria GFP.The sequences of the above wild type proteins and cDNAs encoding thesame are provided in FIGS. 1 to 9.II. amFP486 Characterization and Mutants Thereof (NFP-1; AmCyan)A. Construction of amFP486 Mutants

Two mutants of amFP486 were generated, Mut15 and Mut32. Mut32 has thefollowing amino acid substitutions relative to the wildtype: K65Maccording to GFP alignment numbering as described in Matz et al., supra,or K68M according to self numbering. Mut15 has the following amino acidsubstitutions relative to the wildtype: K65L according to GFP alignmentnumbering as described in Matz et al., supra, or K68L according to selfnumbering. Compared with wildtype amFP486 nucleic acid sequence, Mut15has the following point mutations: A101G, T129C, AAA 202–204TTG, andC240T. (i.e, A to G at position 101 (numbered from beginning of ATG), Tto C at position 129; AAA to TTG at positions 202–204; C to T atposition 240).

Table 2 lists the spectral properties of Mut15 and Mut32.

TABLE 2 Spectral Properties of the Isolated Mut15 and Mut32 AbsorbanceEmission Maximum Relative NFP Maximum Maximum Extinction QuantumBrightness Species Name nm nm Coeff. Yield * Anemonia Mut15 460 48553,400 0.32 0.78 majano Anemonia Mut32 466 488 36,000 0.42 0.69 majano*relative brightness is extinction coefficient multiplied by quantumyield divided by the same value for A. victoria GFP.In addition, a non aggregating mutant of Mut 32 was prepared, where thismutant had the following substitutions: K6E, K68M as compared to wildtype. The orf nucleotide and amino acid sequences of this mutant areprovided in FIG. 10.Additional mutants of NFP-1 include (according to GFP numbering): Y66H;Y66W; A167M; and A167I.B. Construction and Functional Analysis of Vectors

Mut32 DNA was amplified via PCR and reconstructed to EGFP-N1 backbonewith BamHI and NotI restriction enzyme sites. This vector has the samemultiple cloning sites as EGFP-N1 (Clontech Laboratories, Palo Alto,Calif.).

Functional tests of the generated vectors were performed by transienttransfection in 293 cells. After 24-hour expression, brighterfluorescent intensity and less photobleaching of pCNFPMut32-N1 wereobserved by microscopy when compared with pECFP-N1 side by side.

Mut32 has fast folding and bright fluorescent intensity, which makes ituseful for number of applications. Some fusion proteins were tested,such as PKC-gamma-CNFP. PKC was observed to translocate from cytosol tothe plasma membrane when cells were treated with PMA (phorbol12-myristate 13-acetate).

C. Generation of Destabilized amFP486 Vectors as Transcription Reporters

Three destabilized amFP486 vectors were constructed by fusing differentmouse ODC degradation domains such as d1, d2 and d376 to the C-terminalof wild type amFP486 as described in U.S. Pat. No. 6,306,600; thedisclosure of which is herein incorporated by reference. The vectorswere constructed in the EGFP-N1 backbone.

Vectors of pCRE-d1CNFP and pNF-κB-d1CNFP were constructed by placingd1CNFP downstream of cAMP response element (CRE) or NF-κB responseelement, respectively. Expression of d1CNFP is up-regulated uponactivation of these response elements.

D. Functional Analysis of Destabilized amFP486

Functional tests of the destabilized amFP486 were performed by transienttransfection in 293 cells. After 24-hour expression, the fluorescentintensity was decreased gradually from d2, d1 and d376 because of thefusion with different mouse ODC degradation domains. After 4-hourtreatment with protein synthesis inhibitor cycloheximide, d2 fluorescentintensity did not change very much; however, d1 fluorescent intensitydecreased further 50% of its original intensity. The half-life of d1 isaround 4 hours.

MODCd1 is a valuable tool for application as a transcription reporter.However, compared with EGFP-d1 (1-hour half-life), pCNFP-MODCd1half-life (4 hours) is still long, so further mutagenesis for MODCdegradation domain is still needed for shorter half-life version.

Functional tests of vectors pCRE-d1CNFP and pNF-κB-d1CNFP were performedby transient transfection in HEK 293 cells. 16 hours post transfection,10 μm forskolin was added to induce CRE and 100 ng/ml TNF-alpha wasadded to induce NF-κB for 6 hours. Expression of d1CNFP was analyzedusing FACS Calibur. Up to 7 fold increase of fluorescence in forskolininduced CRE activation and 4 fold increase of fluorescence in TNF-αinduced NF-KB activation was observed.

E. Construction and Functional Test for Humanized Mut32 (phCNFP-N1)

Since mammalian expression is a very popular tool, a human favored codonversion of this mutant is needed for better expression in mammaliancells. To generate humanized Mut32, the Mut 32 sequence was firstchanged to human favored codon and 23 oligos (12F and 11R) weredesigned. Next, four rounds of PCR amplification were performed, eachround for 20 cycles. PCR cycle was designed as follows: 94° C. for 1min; 94° C. for 1 min; 40° C. for 1 min; and 72° C. for 1 min. The fourrounds were: for 1^(st) round, mixing 2 μl each of every 4 oligos (60bp), 5 μl buffer, 1 μl pfu, 1 μl dNTP to make total volume of 50 μl.After 20 cycles of PCR, 5 sets of 150 bp and 1 set of 4 last oligos of90 bp products were obtained. For 2^(nd) round, mixing new crude PCRproducts 10 μl each, 5 μl buffer, 1 μl pfu, 1 μl dNTP to make totalvolume of 50 μl. After 20 cycles of PCR, 2 sets of 270 bp and 1 set of210 bp PCR products were obtained. For 3^(rd) round, mixing new crudePCR products. After 20 cycles of PCR, 1 set of 510 bp and 1 set of 450bp products were obtained. For 4^(th) round, mixing new crude products.After 20 cycles of PCR, final PCR product (690 bp) was obtained. FurtherPCR amplification was performed using 1F and 11R primers. As a result,humanized Mut32 was generated. This humanized Mut32 was constituted intoEGFP-N1 backbone.

F. Expression of Wildtype and Mutant amFP486 in Mammalian Cells

The original plasmid amFP486 DNAs (wildtype, Mut15 and Mut32 in pQE30)were used to construct N1 version of amFP486 wildtype, Mut15 and Mut32as described above. The DNAs were inserted into E.coli DH5α. HEK 293cells were transferred with each of the three N1 constructs usingCalcium Phosphate method (Clontech product #K2051–1).

The fluorescent intensity of the transfected cells was analyzed on FACSusing FL1 (510/30) detecting channel. Five samples were analyzed inparallel for each construct. The observed mean value of FL1 fluorescentintensity of the M1 population of each sample is summarized in Table 3.It shows that the average of the mean value of each construct (Wildtype,Mut15, and Mut32) has no significant difference.

TABLE 3 FL1 Fluorescent Intensity of M1 Population Sample # WildtypeMut15 Mut32 1 82.84 106.95 84.51 2 77.52 108.73 91.41 3 111.85 97.0891.30 4 113.06 90.16 98.16 5 104.95 86.34 111.44 Mean 98.04 97.85 95.36G. Generation and Expression of Fusion Protein Mut15-mdm2

The Mut15-mdm2 fusion was generated by the following steps: first, mdm2DNA was obtained by amplifying human Marathon cDNA library (Burke'sLymphoma) using primers:

-   ATGTGCMTACCAACATGTCTGTACC (SEQ ID NO: 19) and CTAGGGGAAATMGTTAGCAC    (SEQ ID No: 20); secondly, the purified PCR product was then    amplified with primers:-   GGAATTCCAGCCATGGTGTGCAATACCAACATGTCTGTACC (SEQ ID NO: 21) and    TCCCCCGGGGGGAAATAAGTTAGCAC (SEQ ID NO: 22)    in order to add Kozac sequence and restriction sites; thirdly, the    purified PCR product from step 2 was digested with EcoR I and Sma I    and inserted into EcoR I and SmaI of NFP1Mut15-N1 vector (this    vector was generated using BamH I and Not I sites of the pEGFP-N1    backbone). The generated Mut15-mdm2 fusion was then expressed in    HEK293 cells.    H. Comparison of the Protein Fluorescent Intensity

PQE30 amFP486 wildtype, Mut15 and Mut32 were transformed into DH5α. Thebacteria grew in the presence of 1 mM IPTG overnight to induce theprotein expression. Cells were lysed in 100 mM Tris, pH 8.0 bysonication. Cell lysate was collected after centrifuge at 3000 rpm for15 minutes at room temperature. The proteins were purified with TALON™Metal Affinity Resin (Clontech Laboratories, Inc., Palo Alto, Calif.).Briefly, after the protein was absorbed on the resin, the beads werewashed in stepwise with first wash, then first elution (50 mM imidazole)and second elution (200 mM imidazole) in 100 mM Tris-HCl, pH 8.0. Theprotein is found mostly in the second step elution. It was found thatMut32 has the highest bacterial expression level, while Mut15 has thelowest.

Samples of each elution fraction were run on SDS-PAGE to check thepurity of the proteins. Both wildtype amFP486 and Mut32 show a singleband, while Mut15 has two more minor bands with higher molecular weight(data not shown).

The protein concentration (fractionII-2) was checked and measured byBradford assay (Bio-Rad standard assay) using BSA as a standard. Thefluorescence intensity (fraction II-2) was determined with a LS50BLuminescence Spectrometer LS50B. EX=458 nm, EM=492 nm, both slits=2.5nm. Table 4 shows the protein concentration, relative fluorescent (FL)intensity and intensity/μg protein in 700 μl volume. It shows that Mut32is as bright as wildtype, while Mut15 is worse than the wildtype.

TABLE 4 Protein Relative FL Intensity/μg Protein Concentration Intensityin 700 μl Volume Wildtype II-2 1.26 μg/5 μl 37.805/5 μl 30.00 Mut15II-20.64 μg/5 μl 10.152/5 μl 15.86 Mut32II-2 6.17 μg/5 μl 186.474/5 μl 30.22III. Characterization of cFP484 and Mutants Thereof (NFP-2)A. Mutant Generation

Two deletion mutants were generated by two separate PCR reactions: Δ19cFP484 lacks the N-terminal first 19 amino acids of cFP484, and Δ38cFP484 lacks the N-terminal first 38 amino acids of cFP484. Mammalianexpression vectors containing the DNA encoding the fluorescent proteinΔ19 cFP484 or Δ38 cFP484 are generated, which are named as pΔ19 NFP2-N1and pΔ38 NFP2-N1, respectively.

B. Transient Expression of Deletion Mutants of cFP484 in Mammalian Cells

HeLa cells were transiently transfected with mammalian expression vectorpΔ19 NFP2-N1 which contains the DNA encoding the fluorescent protein Δ19cFP484. After transfection, cells were incubated for 48 hours at 37° C.then fixed in 3.7% formaldehyde. Cells were mounted in mounting mediumand observed by fluorescence microscopy. Digital images were taken withMetaMorph software (Universal Imaging Corp.) using a monochrome cooledCCD camera (Roper Scientific). The filter set XF 114 (Omega Optical) wasused to visualize fluorescence emitted by Δ19 cFP484. The image waspseudocolored. Δ38 cFP484 is also fluorescent when expressed in HeLacells.

IV. Characterization of zFP506 and Mutants Thereof (NFP-3; ZsGreen)

A. Mutant generation

A mutant of zFP506 was generated, N66M (N65M if numbered according toGFP homology alignment). Compared with wild type zFP506, N66M has themutation of from “AAC” to “ATG” which results in the corresponding aminoacid change from Asparagine (N) to Methionine (M) at the position of 66.The spectral properties of N66M are listed in Table 5.

TABLE 5 Spectral Properties of the Isolated N66M Relative AbsorbanceEmission Maximum Bright- NFP Maximum Maximum Extinction Quantum nessSpecies Name Nm nm Coeff. Yield * Zoanthus N65M 496 506 62,000 0.63 1.78sp. *relative brightness is extinction coefficient multiplied by quantumyield divide by the same value for A. victoria GFP.The following additional mutants were also generated: FP3-NA: K5E, K10E,N66M (non-aggregating mutant)(See FIG. 11); Yellow (A64G, N66K,N69D)(See FIG. 12); Yellow/bright (A64G, N66K, N69D, D94N, M120V, K157R,P231S) (See FIG. 13).Additional mutants of NFP-3 include (according to GFP numbering): M167A;M167H; and N65K/N68D.B. Construction and Functional Analysis of Vectors

Non-humanized zFP506 DNA was amplified via PCR and reconstructed intoEGFP-N1 backbone. This vector has the same multiple cloning sites asEGFP-N1. Functional test of the generated vector was performed bytransient transfection in 293 cells. 24 hours post transfection,expression of zFP506 was examined under fluorescent microscope. zFP506showed good fluorescent intensity and comparable to EGFP-N1.

C. Generation of Destabilized zFP506 Vectors as Transcription Reporters

Since zFP506 is very stable, it is necessary to generate destabilizedversions of zFP506 in order to observe the rapid turnover of theprotein. By using the same technology for destabilized EGFP, twodestabilized zFP506 vectors were constructed by fusing mouse ODCdegradation domain to the C-terminal of zFP506. The d1 version ofdestabilized zFP506 has three E to A mutations within MODC degradationdomain comparing to d2 version, therefore result in a shorter half-lifeof the protein to which MODC degradation domain fused to. Destabilizedd1zFP506 and d2zFP506 were constructed in EGFP-N1 backbone

D. Functional Analysis of Destabilized zFP506

Wildtype d1zFP506 was transiently transfected into 293 cells. 24 hoursafter transfection, CHX was added to stop protein synthesis. After 4hour treatment, cells were examined under fluorescent microscope. Itshows that fusion of MODC domain to the zFP506 slightly decreases thefluorescent intensity compared to zFP506 itself. After 4 hour treatment,there is 50% fluorescent intensity decrease.

E. Application of Destabilized d1zFP506 as Transcription Reporters

Destabilized d1zFP506 was constructed into pCRE-d1GNFP and pNF-κB-d1GNFPvectors. Its expression was regulated under cAMP response element (CRE)or NF-κB response element, respectively. These vectors were transientlytransfected into 293 cells, and 24 hours post transfection, theexpression of d1GNFP was induced by Forskolin or TNF-α. 6 hours afterinduction, the culture was analysed by FACS. CRE-d1GNFP showed 7 fold ofinduction in fluorescence intensity, while 4 fold of induction wasobtained in NF-κB-d1GNFP (data not shown). This demonstrated that thedestabilized form of GNFP is applicable as transcription reporters.

F. Construction and Functional Test for Humanized zFP506 and HumanizedN66M

Since mammalian expression is a very popular tool, human favored codonversion is needed for better expression in mammalian cells. Each pieceof human favored codon oligos was linked to form the full length of wildtype and/or mutant zFP506 (hGNFP-zFP506; hGNFP-N65M. This humanizedzFP506 was constituted into EGFP-N1 backbone.

V. Characterization of zFP538 and mutants thereof (NFP-4; ZsYellow)

A mutant of zFP538 M129V (as measured from the start of the protein) wasgenerated. M129V (M128V using GFP numbering) was generated byintroducing a wrong nucleotide in PCR during site-specific mutagenesisat position 65. One bright yellow colony was obtained, and the sequenceof this clone was performed. It showed that this clone contained wildtype amino acid Lysine (K) at position 65, but had a substitution fromMethionine (M) to Valine (V) at position 129 (numbering from start ofprotein; at position 128 if numbering according to GFP homologyalignment).

Further investigations showed that M129V has spectral characteristicsvery similar to wild type protein zFP538 but folds much faster. Table 6lists the spectral properties of M129V.

TABLE 6 Spectral Properties of the Isolated M128V Absorbance EmissionMaximum Relative NFP Maximum Maximum Extinction Quantum BrightnessSpecies Name nm nm Coeff. Yield * Zoanthus M128V 531 540 25,360 0.430.50 sp. *relative brightness is extinction coefficient multiplied byquantum yield divided by the same value for A. victoria GFP.The following additional mutants were also generated: FP4-NA (K5E, K9T,M129V)(Non-Aggregating Mutant); Green (K65M GFP numbering; K66M selfnumbering).Additional mutants of NFP-1 include (according to GFP numbering): D68N.A. Construction and Functional Analysis of Vectors

Both wildtype (wt) and mutant zFP538 DNA were amplified via PCR andreconstructed to EGFP-N1 backbone. This vector has the same multiplecloning sites as EGFP-N1. Both pYNFPwt and pYNFPW129V keep the samemultiple cloning sites as EGFP-N1. Functional test of the generatedvectors was performed by transient transfection in 293 cells. After24-hour expression, pYNFPwt, pYNFPM129V and EYFP were compared side byside: pYNFPwt showed less fluorescent intensity than EYFP (data notshown); however, pYNFPM129V showed as bright fluorescent intensity asEYFP by fluorescent microscopy.

B. Generation of Destabilized zFP538 Vectors as Transcription Reporters

By using the same technology for destabilized EGFP, destabilized zFP538vectors were constructed by fusing different mouse ODC degradationdomains such as d1 and d2 to the C-terminal of zFP538. The d1 version ofdestabilized YNFP has three E to A mutations within MODC degradationdomain compared to d2 version. Vectors pYNFPM128V-MODCd1 andpYNFPM128V-MODCd2 were constructed in EGFP-N1 backbone.

C. Functional Analysis of Destabilized zFP538

Functional test of the destabilized zFP538 was performed by transienttransfection in 293 cells. After 24-hour expression, the fluorescentintensity was decreased gradually from d2 and d1 because of the fusionwith different mouse ODC degradation domains. After 4-hour treatmentwith protein synthesis inhibitor cycloheximide, d2 fluorescent intensitydid not change very much; however, d1 fluorescent intensity decreasedfurther 50% of its original intensity. The half-life of d1 is around 4hours.

M129V has fast folding and bright fluorescent intensity, which makes ituseful for number of applications. Some fusion proteins were tested suchas PKC-gamma-YNFP (M129V). PKC-gamma was observed to translocate fromcytosol to the plasma membrane when cells were treated with PMA (Phorbol12-Myristate 13-Acetate).

D. Construction and Functional Test for Humanized M129V

Humanized M129V was generated, and then placed into the pEGFP-N1backbone. This vector has the same multiple cloning sites as pEGFP-N1.Construction of C1 and PEGFP is in the process.

E. Structural Characterization of Green Mutant

1. Analytical Ultracentrifugation:

Data show zFP538 is non-dissociating trimer of 80 kD/dimer-tetramerequilibrium with S values of about 6 and 8; results not conclusive, butsure that there is more than one species.

2. Structure:

Tetramer, extremely similar to DsRed; no idea why wildtype is yellow,chromophore is identical to that of GFP; although the tetramers of dsRedand zFP538 are essentially identical in the overall configuration and inthe positioning of the interfacial regions, the residues involved ininterfacial contacts are not conserved. This finding indicates that alot of variability is tolerated and monomers can be created.

VI. Characterization of dsFP483 and Mutants Thereof (NFP-5)

Mutants of NFP-5 include (according to GFP numbering): N68S; 1112S; andN68S/1112S.

VII. Characterization of drFP583 and Mutants Thereof (NFP-6; DsRed;DsRed2)

A. Expression in Mammalian Cells

HeLa cells were transfected either with plasmid pDsRed1-N1 (vectorcontaining the DNA encoding drFP583) or plasmid pEGFP-C1 (encoding EGFPfrom Aequorea victoria). Immediately after the transfection, cells weremixed and plated on coverslips. Cells were incubated for 48 hours at 37°C. then fixed in 3.7% formaldehyde. Cells were mounted in mountingmedium and observed by fluorescence microscopy. Images were taken fromthe same field of view with Chroma filter set 31001 for EGFP and filterset 31002 for drFP583 using a cooled CCD camera (Roper Scientific) andMetaMorph software (Universal Imaging). The images were pseudocoloredand overlayed. Phase contrast was taken from the same field of view andoverlayed.

B. Generation of Humanized drFP583

Since mammalian expression is a very popular tool, human favored codonversion is needed for better expression in mammalian cells. HumanizeddrFP583 was therefore generated by changing wild type drFP583 nucleotidesequence to optimize the codons for expression of the fluorescentprotein. The nucleotide sequence of this humanized mutant is provided inFIG. 16.

C. Expression of Humanized drFP583 in Mammalian Cells

HeLa cells were transiently co-transfected with plasmids pECFP-Nuc,pEYFP-Tub and pDsRed1-Mito (humanized drFP583). After transfection,cells were incubated for 48 hours at 37° C. then fixed in 3.7%formaldehyde. Cells were mounted in mounting medium and observed byfluorescence microscopy. Images were taken of one cell co-expressing allthree fluorescent proteins with Omega filter set XF 35 for DsRed1-Mito,XF 104 for EYFP-Tub and XF 114 for ECFP-Nuc using a cooled CCD camera(Roper Scientific) and MetaMorph software (Universal Imaging).Individual images were pseudocolored and overlayed to show all threesignals in one image. Protein DsRed1-Mito localizes to mitochondria,EYFP-Tub localizes to the microtubular network, and ECFP-Nuc localizesto the nucleus.

As a conclusion, drFP583 does emit to a low extent also in the cyan(ECFP), green (EGFP) and yellow-green (EYFP) emission channels (filtersets). High expression levels or highly concentrated protein inintracellular structures can therefore result in high signal intensitiesthat will give some bleedthrough in the other emission wavelengths. Thebleedthrough is small and should not affect multiple labeling recordingin most cases.

D. Mutants of Humanized drFP583

Mutants of humanized drFP583 were generated using error prone PCRtechnique (Clontech). Mutations occurred at amino acids 42, 71, 105,120, 161 and 197 (numbering starting from the first Methionine). Table 7lists the mutants that were generated and their properties.

TABLE 7 Mutants of Humanized drFP583 Mutant Mutations Properties E5V105A, S197T Overnight in E. coli emitting green fluorescence; in vitromaturing to red over 28 h at 37° C. on 80% (retains 20% green peak);folding faster than wild type drFP583 (~28 h) E8 N42H Always two peaksgreen & red (~1:1) folding faster than E5 (~8 h) E5up V105A red from thebeginning; folding faster than E5 (~12 h) E5 S197T phenotype is similarto E5 down E57 V105A, like E5 but folding faster (~8–10 h); I161T, S197A~5% of green peak left at the end (See FIG. 18) AG4 V71M, V105A, brightgreen, no red at all; fast folding S197T (~16 h) AG45 V71M, V105A, likeAG4 but twice brighter Y120H, S197T FP6 R2A, K5E, K9T, Non-aggregating(See FIG. 19) (E57)- V105A, I161T, NA: S197A. E5-NA: R2A, K5E, K9T,(non-aggregating Fluorescent Timer) V105A, S197T (See FIG. 17) E83 N42H,V71A, I180VE. Characterization and Applications of E5 Mutant

E5 (V105A, S197T) changes its fluorescence from green to red overtimeboth in vitro and in vivo, in E. coli and in mammalian cells. Also, E5folds faster than wild type drFP583 both in E. coli and mammalian cells.

Since it allows the “two color” reporting mode for monitoring of thepromoter activity, i.e., for both active or shutdown state of thepromoter, there is a separate color, serving as an indicator of thatstate, E5 can be used as a transcriptional reporter. Different from “onecolor” mode, “two color” mode has a measurable signal (color) presentfor both states of the process as opposed to “one color” mode (e.g.destabilized GFP) wherein the absence of the color is an indicator ofthe second state. Namely, newly produced E5 protein fluoresces in green,indicating on-going promoter activity. Over time, the protein willmature, acquiring the red fluorescence. So if the promoter is no longeractive, all the protein will eventually mature, resulting in thedominant red fluorescence. In case the promoter is still active both redand green fluorescence will be readily detected. Thus E5 as a “twocolor” reporter allows study of gene expression similar to destabilizedGFP, but with permanent “signature” of past gene activity in the cells,tissues or entire organism. For example, at the tissue level, E5 mayhelp to distinguish the stem cells from differentiated cells. Providingthe promoter is only active in the stem cells, the E5 reporter willlabel the stem cell population in green and red, the progenitor cellswould be labeled predominantly in red, the terminally differentiatedwill not fluoresce (due to the titration out of protein during celldivision).

E5 can be used for spatial and temporal visualization of newlysynthesized vs. accumulated fusion proteins. That is, E5 could functionlike a fusion tag. Possible applications envisaged at differentorganizational levels. At the cellular level, E5 may help to visualizeand distinguish the newly synthesized proteins in various compartmentssuch as outer membrane, microvillae, ER, Golgi, mitochondria, nuclei,various components of cellular matrix and focal adhesion complexes. Atthe tissue level, E5 may be helpful in visualizing newly formed vs.preexisting structures e.g. membrane junctions, components ofextracellular matrix.

One of the most fascinating applications of E5 seems to be in the studyof the mother-daughter relationship during the cell division andmigration. A wide horizon is opening in the fields of development aswell as in the adult organisms to study the cell migration anddifferentiation. Allowing visualization of the expression “history”, E5can help to distinguishing between the mother cells where the promoteris actually active vs. the daughter cells containing the accumulatedprotein but not producing fresh protein anymore. It would enable thestudy of the cell fates during development and organ remodeling, thusdistinguishing between cell migration and cell expansion ordifferentiation.

In conclusion, E5 is basically applicable to any situation where GFP waspreviously used. Main advantage is that E5 can track down “the history”of promoter activity or protein localization in cells or tissues. With abetter protein stability than GFP, E5 allows longer analysis window(wild type drFP583 is stable for at least 4 weeks in Xenopus, while EGFPstarts to faint after two weeks).

F. Characterization and Applications of E8 Mutant

E8 (N42H) has two fluorescence maximums, green and red, at all times andit folds much faster than drFP583 (Table 7).

Since it detects both green and red fluorescence simultaneously, E8 maybe useful for studying processes related to blood circulation andproteins/cells trafficking. Blood absorbs the green fluorescence; thusonly the red fluorescence will be visible while the protein istrafficking in the blood. Both green and red fluorescence could bedetected outside the bloodstream making the whole process easy tovisualize and record. Monitoring both red and green fluorescencesimultaneously may also help to reduce the background fluorescenceproblems for some tissues or cells.

G. Generation of drFP583/dmFP592 Hybrid Using Shuffling Procedure

Non-humanized wild type coding region fragments from drFP583 and dmFP592were amplified by PCR (22 cycles, 95° C., 15 sec., 68° C. 1 min 20 sec.)using 1 ng of corresponding bacterial expression plasmids (pQE-30derivatives with drFP583 and dmFP592 inserts, respectively) astemplates. Oligonucleotides

-   A (ACATGG ATCCAGGTCTTCCAAGAATGTTATC, SEQ ID NO: 23),-   B (TAGTACTCG AGCCAAGTTCAGCCTTA, SEQ ID NO: 24),-   C (ACATGGATCCAG TTGTTC CAAGAATGTGAT, SEQ ID NO: 25), and-   D (TAGTACTCGAGGCCATTA CCGCTAATC, SEQ ID NO: 26)    were used as primers for amplifying these fragments in a    concentration of 0.2 mM.

The PCR products were then purified by QIAquick PCR Purification Kit(QIAGEN). Afterwards, the purified fragments drFP583 and dmFP592(300–500 ng each) were digested with restriction endonucleases EcoRI,HindIII and DraI (10 U each) simultaneously. Reactions were performed inBamHI restriction buffer (NEB) supplemented BSA for 3 h at 37° C. Totalreaction volume was 30 ml. Upon completion, the resulted restrictionfragments from each restriction reaction were separated byelectrophoresis in agarose gels (1.5%), cut from gel and purified byQIAquick Gel Purification Kit (QIAGEN). The resulting sets of thepurified restriction fragments from both drFP583 and dmFP592 werecombined together and 50 ng of them were put into ligation mix (1× T4DNA ligation buffer, 400 NEB U of T4 DNA ligase) in total volume of 30ml. The ligation was performed for 3 h at room temperature and stoppedby heating at 70° C. within 20 min.

The ligation mixture was then diluted by water ten-folds, and 1 ml ofthe dilution was preformed for PCR reaction (20 cycles, 95° C., 15 sec.68° C. 1 min 20 sec) as template. Four oligonucleotides A, B, C, and D(SEQ ID NOS: 58–61, respectively) were used simultaneously as primersfor amplifying these fragments in a concentration of 0.1 mM each. Afterelectrophoresis in an agarose gel (1.5%), the target fragment waspurified by QIAquick Gel Purification Kit (QIAGEN) and digested withrestriction endonucleases BamHI and XhoI (30–50 U each) simultaneouslyin BamH I restriction buffer (NEB) supplemented with BSA for 3 h at 37°C. After purification, the resulting fragment was cloned in pQE-30plasmid linearized by BamHI and SalI. Ligation reaction was performed in1× T4 DNA ligation buffer and 400 NEB U of T4 DNA ligase with a totalvolume of for overnight at 16° C. After transformation of E.coli cellsby ⅕ of the ligation volume and incubation on LB/1% agar plates whichwere supplemented by 100 mg/ml Ampacilin and 0.1 mM IPTG at 37° C. forovernight, the resulting E.coli colonies were screened visually underfluorescent microscope using rhodamine filter set. The brightest redcolonies were picked up and placed in 200 ml LB medium with 100 mg/ml ofAmpacilin. At OD₆₀₀=0.6, the E.coli culture was induced by IPTG (finalconcentration was 1 mM) and the fermentation continued for overnight.Purification of recombinant protein containing N-terminus 6× his tag wasperformed using TALON metal-affinity resin according to manufacturer'sprotocol.

H. Spectral Properties of drFP583/dmFP592 Hybrid

The emission and excitation spectra for drFP583/dmFP592 hybrid proteinare basically the same as for dmFP592. Table 8 lists the spectralproperties of drFP583/dmFP592 hybrid protein.

TABLE 8 Spectral Properties of drFP583/dmFP592 Hybrid AbsorbanceEmission Maximum Relative Relative nFP Maximum Maximum ExtinctionQuantum Brightness Name nm nm Coeff. Yield* ** drFP583/ 573 592 35,0000.24 0.3 dmFP592 *relative quantum yield was determined as compared tothe quantum yield of A. victoria GFP. **relative brightness isextinction coefficient multiplied by quantum yield divided by the samevalue for A. victoria GFP.I. Humanized drFP583/dmFP592 Hybrid and Mutants

drFP583/dmFP592 hybrid was humanized. Further, two mutants weregenerated based on the humanized drFP583/dmFP592, i.e.,drFP583/dmFP592-2G and drFP583/dmFP592-Q3. drFP583/dmFP592-2G (i.e.6/92G) contains two substitutions, K15Q and T217S (Self numbering). Thismutant was derived from the humanized drFP583/dmFP592 hybrid gene byrandom mutagenesis using Diversity PCR Mutagenesis Kit (Clontech)according to the corresponding protocol. drFP583/dmFP592-Q3 (6/9Q)contains three substitutions, K15Q and K83M and T217S (self numbering).drFP583/dmFP592-Q3 mutant was derived from drFP583/dmFP592-2G mutant byrandom mutagenesis using Diversity PCR Mutagenesis Kit (Clontech)according to the corresponding protocol. A non-aggregating mutant of 6/9Q, i.e. 6/9 QNA, was also produced, which the following substitutions:K5E, K9T, K15Q, K83M, T217S (self numbering). The sequences of ahumanized 6/9 Q hybrid protein are provided in FIG. 22.

drFP583/dmFP592-2G has similar brightness and folding rate as fornon-humanized drFP583/dmFP592 hybrid. While drFP583/dmFP592-Q3 could beseen in E.coli cells as more dark red than parental variant, i.e.,drFP583/dmFP592-2G, and the purified protein solution has purple color.drFP583/dmFP592-Q3 has the emission maximum of 616 nm and excitationmaximum of 580 nm.

J. Applications of Hybrid Mutants

Similar to fluorescent protein drFP583 or dmFP592, drFP583/dmFP592-Q3can be used as a tool for investigation of protein expression, transportand protein interactions in vivo, monitoring of promoter activity, andas a transcription reporter or fusion tag. Besides, drFP583/dmFP592-Q3can be chosen as the most convenient partner to one of the existinggreen fluorescent protein variants in two/triple color labeling assaysfor simultaneous detection of expression of two or more proteins in vivodue to its strongly red-shifted position of emission maximum andpractical absence of excitation in green part of spectrum except anyspectral overlapping and background fluorescence.

The method of generating drFP583/dmFP592 hybrid can have a generalutility for generating hybrid genes (i.e., genes containing parts ofdifferent genes in various combinations) with improved fluorescentcharacteristics.

Additionally, drFP583/dmFP592-Q3 is the first red-shifted mutant, whichdemonstrates that spectral-shifted mutant could be obtained by randommutagenesis.

K. Further Characterization.

Parental residual green in vitro Mutant Position of clone/ fluorescence(% maturation name mutation(s) gene from red) t½ DsRed wild type 4–5after 18–24 Not seen ON in 9–10 hours E. coli, 16–24 h in 293 cells E5V105A,S197T, FP#6 20% after 43 In E. coli 9–10 (w.t.) hours, eventuallyGREEN o/night, drops to 10% in 293 cells both color develop faster thanwt E5-UP V105A E5(split) 3% after 24 hours beeter folding in 9–10eventually zero bacteria E5-DOWN S197T E5(split) similar to E5 similarto E5 9–10 E57 V105A,I161T, E5 5% after 8 h, 4% better folding 3–4 S197A after 24 hours and faster maturation of fluorophore AG4 V71M, E5100% green at all bright GREEN, 1.5–2   V105A,S197T time no RED at allAG45 V71M,V105A, AG4 100% green at all ~2x brighter 1.5–2   Y120H,S197Ttime than AG4 E8 N42H FP#6 Green/Red ratio better folding 3–4  (w.t.) is3:2 and faster maturation of fluorophore E83 N42H,V71A, E8 Green/Redratio same as E8 3–4  I180V is 1:1 green fluorescence: RFP about 4–5% wtafter 18–24 hours, remains. E5 about 20% after 43 hours eventually dropsto 10% E5 up about 3% after 24 hours eventually goes to zero. E5 downabout 20% after 24 hours, eventually drops to 10% E57 5% after 8 hours,4% after 24 hours; faster maturation than wt RFPVIII. Characterization of asFP600 and Mutants Thereof (NFP-7; AsRed)A. Mutant Generation

A mutant of asFP600 was generated, Mut1. Compared with wild typeasFP600, Mut1 has the following substitutions: T68A and A143S from startof protein (T to A at position 70 and A to S at position 148 undernumbering according to GFP). Target substitution A143S was generated bymeans of site-specific mutagenesis using PCR with primers that carriedthe mutation. During this mutagenesis random substitution T68A wasgenerated by introducing a wrong nucleotide in PCR. The substitutionT68A is not necessary for fluorescence and practically does not affectthe fluorescence. Table 9 lists the spectral properties of Mut1. Anothermutant of asFP600 was generated, having a substitution of the Alanine atposition 184 to Serine (according to GFP numbering).

TABLE 9 Spectral Properties of the Isolated Mut1 Absorbance EmissionMaximum Relative NFP Maximum Maximum Extinction Quantum BrightnessSpecies Name Nm nm Coeff. Yield * Anemonia Mut1 573 595 15,500 0.05 0.03sulcata *relatlve brightness is extinction coefficient multiplied byquantum yield divided by the same value for A. victoria GFP.Yet one more mutant was generated, i.e., mut M35-5/mut1 (=7A): F4L,K12R, F35L, T68A, F84L, A143S, K163E, M202L (positions relative to thestart of the protein)A non-aggregating version of this mutant was also made, i.e., 7A-NA:K6T, K7E, F4L, K12R, F35L, T68A, F84L, A143S, K163E, M202L (positionsrelative to the start of the protein)The humanized FP-7 sequences are provided in FIG. 21.B. Construction and Functional Analysis of Vectors

Non-humanized mutant asFP600 (RNFP) DNA were amplified via PCR andreconstructed to EGFP-N1 backbone. This vector (pRNFP-N1) has the samemultiple cloning sites as EGFP-N1.

Functional test of the generated vector was performed by transienttransfection in 293 cells. 24 hours post transfection, expression ofasFP600 was examined under fluorescent microscope. asFP600 showed goodfluorescent intensity, however, the expression of asFP600 concentratedat the nucleus.

C. Generation of Cytosal Expressed asFP600

Since the nuclear localization of asFP600 limited some of theapplication of this protein as transcription reporter or pH sensor,cytosal expression of this protein would be needed for this purpose. Anuclear export sequence in humanized codon usage was fused to theN-terminus of asFP600, and placed into the EGFP-N1 vector, resulted inpNE-RNFP.

Functional test of NE-RNFP is performed by transient transfect thepNE-RNFP into 293 cells. 24 hours post transfection, expression ofNE-RNFP is examined under fluorescence microscope. Red fluorescence wasobserved to be distributed in the cytosol but not in the nucleus.

D. Generation of Destabilized asFP600 Vectors as Transcription Reporters

Since asFP600 is very stable, it is necessary to generate destabilizedversions of asFP600 in order to observe the rapid turnover of theprotein. By using the same technology for destabilized EGFP, twodestabilized NE-RNFP vectors were constructed by fusing mouse ODCdegradation domain to the C-terminal of NE-RNFP. The d1 version ofdestabilized RNFP has three E to A mutations within MODC degradationdomain comparing to d2 version, therefore result in a shorter half-lifeof the protein to which MODC degradation domain fused. Destabilized d1RNFP and d2RNFP were constructed in EGFP-N1 backbone.

E. Functional Analysis of Destabilized asFP600

d2 version of the none-humanized asFP600 was transiently transfectedinto 293 cells. One day after transfection, CHX was added to inhibitprotein synthesis. 3 hours after treatment, cells were examined underfluorescent microscope. It showed that fluorescent intensity decreased˜50%.

F. Construction and Functional Test for Humanized Mut1

Humanized Mut1 was generated. The humanized Mut1 was then placed intothe pEGFP-N1 backbone. This vector has the same multiple cloning sitesas pEGFP-N1. Construction of C1 and pEGFP is in the process.

It is evident from the above discussion and results that the subjectinvention provides important new chromoproteins and fluorescent proteinsand nucleic acids encoding the same, where the subject proteins andnucleic acids find use in a variety of different applications. As such,the subject invention represents a significant contribution to the art.

IX. Summary Table of Mutant Fluorescent Proteins

TABLE 10 Mutants: Mutants: aa numbering according to GFP aa numberingaccording to aa NFP# name homology alignment sequence of protein proper1 amFP486 K65M (mut32) K68M (mut32) K65L (mut15) K68L (mut15) FP1-NA:K6E, K65M FP1-NA: K6E, K68M (non-aggregating mut 32) (non-aggregatingmut 32) 2 CFP484 19 (N-terminal 19 aa deleted) 38 (N-terminal 38 aadeleted) 3 zFP506 Mut: N65M Mut: N66M FP3-NA: K5E, K10E, N65M (non-FP3-NA: K5E, K10E, N66M (non- aggregating mutant) aggregating mutant)Yellow (A63G, N65K, N68D) Yellow (A64G, N66K, N69D) Yellow/bright (A63G,N65K, N68D, Yellow/bright (A64G, N66K, N69D, D95N, M119V, K158R, P232S)D94N, M120V, K157R, P231S) 4 zFP540 Mut: M128V Mut: M129V FP4-NA: K5E,K9T, M128V (non- FP4-NA: K5E, K9T, M129V (non- aggregating mutant)aggregating mutant) Green (K65M) Green (K66M) 5 dsFP483 6 DsFP583 FP6(E57)-NA: R2A, K5E, K9T, V105A, I161T, S197A. E5 (V105A, S197T;Fluorescent Timer) E5-NA: NA: R2A, K5E, K9T, V105A, S197T(non-aggregating Fluorescent Timer); E5up (V105A; faster maturing,brighter) E5down (S197T, fluorescent timer phenotype) E57 (V105A, I161T,S197A; faster chromophore maturation, brighter) AG4 (V71M, V105A, S197T;green only) AG45 (V71M, V105A, Y120H, S197T; green only) E8 (N42H;green/red) E83 (N42H, V71A, I180V; green/red) 6/9 6/9 Q (K15Q, K83M,T217S) hybrid 6/9Q-NA: (S2del, C3del, accidentally deleted) K5E, K9T,K15Q, K83M, T217S. Other mutants: 6/92G (K15Q, T217S) 7 asFP595 mut1:T70A, A148S mut1: T68A, A143S 7A mut M35-5/mut1(=7A): F7L, K15R, mutM35-5/mut1(=7A): F38L, T70A, F88L, A148S, K170E, F4L, K12R, F35L, T68A,F84L, M208L. A143S, K163E, M202L 7A-NA::K6T, K7E, F4L, K12R, F35L, T68A,F84L, A143S, K163E, M202L (non-aggregating version of 7A)

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

1. A nucleic acid present in other than its natural environment, whereinsaid nucleic acid encodes a chromo- or fluorescent protein, wherein saidprotein has a sequence identity of at least 90% with SEQ ID NO:
 12. 2.The nucleic acid according to claim 1, wherein said protein has anabsorbance maximum ranging from about 300 to 700 nm.
 3. The nucleic acidaccording to claim 1, wherein said protein has an absorbance maximumranging from about 350 to 650 nm.
 4. The nucleic acid according to claim1, wherein said protein has an absorbance maximum ranging from about 400to 600 nm.
 5. The nucleic acid according to claim 1, wherein saidprotein has an excitation spectrum ranging from about 300 to 700 nm andan emission spectrum ranging from about 400 to 800 nm.
 6. The nucleicacid according to claim 1, wherein said protein has an excitationspectrum ranging from about 350 to 650 nm and an emission spectrumranging from about 425 to 775 nm.
 7. The nucleic acid according to claim1, wherein said protein has an excitation spectrum ranging from about400 to 600 nm and an emission spectrum ranging from about 450 to 750 nm.8. The nucleic acid according to claim 1, wherein said protein has oneor more amino acid substitutions selected from amino acid substitutionsat positions 2, 5, 9, 105, or 197 as compared to SEQ ID NO:12.
 9. Thenucleic acid of claim 1, wherein said protein has one or more amino acidsubstitutions selected from R2A, K5E, K9T, V105A, and S197T as comparedto SEQ ID NO:12.
 10. An expression cassette comprising: (a) atranscriptional initiation region functional in an expression host; (b)a nucleic acid according to claim 1; and (c) a transcriptionaltermination region functional in said expression host.
 11. An isolatedhost cell comprising an expression cassette according to claim 10 aspart of an extrachromosomal element or integrated into the genome of ahost cell as a result of introduction of said expression cassette intosaid host cell.
 12. A kit comprising the nucleic acid according to claim1 and instructions for using said nucleic acid.
 13. A nucleic acidpresent in other than its natural environment that encodes a chromoand/or fluorescent protein, wherein said protein is either: (a) from anon-bioluminescent Cnidarian species; or (b) from a non-PennatulaceanAnthozoan and wherein said protein has a sequence identity of at least90% with SEQ ID NO:
 12. 14. The nucleic acid according to claim 13,wherein said non-bioluminescent Cnidarian species is an Anthozoanspecies.
 15. The nucleic acid according to claim 14, wherein saidnucleic acid is isolated.
 16. The nucleic acid according to claim 14,wherein said protein has the amino acid sequence of SEQ ID NO:
 12. 17. Anucleic acid present in other than its natural environment that encodesa chromo and/or fluorescent protein that is either: (i) from anon-bioluminescent Cnidarian species; or (ii) from a non-PennatulaceanAnthozoan and wherein said protein has a sequence identity of at least90% with SEQ ID NO: 12, and wherein said protein has an absorbancemaximum ranging from about 300 to 700 nm.
 18. The nucleic acid accordingto claim 17, wherein said non-bioluminescent Cnidarian species is anAnthozoan species.
 19. A construct comprising a vector and a nucleicacid that encodes a chromo and/or Fluoresent protein that is either: (i)from a non-bioluminescent Cnidarian species; or (ii) from anon-Pennatulacean Anthozoan and wherein said protein has a sequenceidentity of at least 90% with SEQ ID NO:
 12. 20. The construct accordingto claim 19, wherein said non-bioluminescent Cnidarian species is anAnthozoan species.